Antibody molecules to dengue virus and uses thereof

ABSTRACT

Antibody molecules that specifically bind to dengue virus are disclosed. In certain embodiments, the antibody molecule bind to dengue virus serotypes DV-1, DV-2, DV-3, and DV-4. The antibody molecules can be used to treat, prevent, and/or diagnose dengue virus.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.61/938,646, filed on Feb. 11, 2014, U.S. Provisional Application No.62/017,970, filed on Jun. 27, 2014, and U.S. Provisional Application No.62/046,379, filed on Sep. 5, 2014. The disclosures of the priorapplications are considered part of (and are incorporated by referencein their entirety in) the disclosure of this application.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jan. 7, 2015, isnamed P2029-700210_SL.txt and is 73,010 bytes in size.

BACKGROUND

Dengue virus is a positive-sense RNA virus belonging to the Flavivirusgenus of the family Flaviviridae. Dengue virus is widely distributedthroughout the tropical and semitropical regions of the world and istransmitted to humans by mosquito vectors. Dengue virus is a leadingcause of hospitalization and death in children in at least eighttropical Asian countries (WHO, 1997. Dengue haemorrhagic fever:diagnosis, treatment prevention and control—2nd ed. Geneva: WHO). Thereare four serotypes of dengue virus (DV-1, DV-2, DV-3, and DV-4) whichannually cause an estimated 50-100 million cases of dengue fever and500,000 cases of the more severe form of dengue virus infection, denguehemorrhagic fever/dengue shock syndrome (DHF/DSS) (Gubler, D. J. &Meltzer, M. 1999 Adv Virus Res 53:35-70). DHF/DSS is seen predominatelyin children and adults experiencing a second dengue virus infection witha serotype different than that of their first dengue virus infection andin primary infection of infants who still have circulatingdengue-specific maternal antibody (Burke, D. S. et al. 1988 Am J TropMed Hyg 38:172-80; Halstead, S. B. et al. 1969 Am J Trop Med Hyg18:997-1021; Thein, S. et al. 1997 Am J Trop Med Hyg 56:566-72).

The different serotypes of dengue virus differ at the amino acid levelby about 25-40% and have antigenic differences, and this variation hashindered efforts to produce a therapy effective against all serotypes.

All four dengue virus serotypes display an E (envelope) protein on theviral surface. The E protein contributes to the attachment of the virusto a host cell. The E protein comprises a DI domain (a nine-strandedbeta-barrel) a DII domain (a domain implicated in fusion with the hostcell), and a DIII domain (an immunoglobulin-like domain). The humoralresponse to E protein in humans generally targets the DI and DIIregions, with much of the antibodies exhibiting high cross-serotypereactivity but low neutralization activity.

There is a need in the art for new prophylactic and therapeutictreatments for dengue virus, and especially for treatments that areeffective against all four serotypes of the virus.

SUMMARY

This disclosure provides, at least in part, antibody molecules that bindto the dengue virus, for example, the dengue virus E protein, and whichcomprise functional and structural properties disclosed herein. In someembodiments, the antibody molecules bind to the “A” beta-strand of EDIII(the E protein DIII domain). In some embodiments, the antibody moleculesbind to and/or neutralize at least 1, 2, 3, or 4 dengue virus serotypes,e.g., DV-1, DV-2, DV-3, and DV-4. In some embodiments, the antibodymolecule is selected from Table 1. In some embodiments, the antibodymolecules comprise a deletion of VH S26 and/or a VH T33V substitutioncompared to antibody A11. These mutations, in some embodiments, mayimprove one or more properties, e.g., improve antibody affinity for oneor more dengue virus serotypes, for example serotype DV-4. In someembodiments, the antibody molecule targets a site on EDIII that isconserved across all four dengue serotypes. Nucleic acid moleculesencoding the antibody molecules, expression vectors, host cells,pharmaceutical compositions, and methods for making the antibodymolecules are also provided. The anti-dengue antibody moleculesdisclosed herein can be used (alone or in combination with other agentsor therapeutic modalities) to treat, prevent and/or diagnose denguevirus, e.g., DV-1, DV-2, DV-3, or DV-4.

Accordingly, in certain aspects, this disclosure provides an antibodymolecule (e.g., an isolated, recombinant, or humanized antibodymolecule) having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or all) of the followingproperties from List 1:

-   -   a) Binds to EDIII (e.g., one or more EDIII from any dengue virus        serotype, e.g., from DV-1, DV-2, DV-3, or DV-4, e.g., all four        EDIII from DV-1, DV-2, DV-3, or DV-4) with high affinity, e.g.,        with a dissociation constant (K_(D)) of less than about 100 nM,        typically about 10 nM, and more typically, about 10-0.01 nM,        about 5-0.01 nM, about 3-0.05 nM, about 1-0.1 nM, or stronger,        e.g., less than about 80, 70, 60, 50, 40, 30, 20, 10, 8, 6, 4,        3, 2, 1, 0.5, 0.2, 0.1, 0.05, or 0.01 nM,    -   b) Binds to DV-4 EDIII with high affinity, e.g., with a        dissociation constant (K_(D)) of less than about 100 nM, e.g.,        about 10 nM, e.g., about 10-1 nM or stronger, e.g., less than        about 10, 8, 6, 5, 4, or 3 nM,    -   c) Binds to DV-4 and/or DV-3 EDIII domain with a greater        affinity than antibody A11 (also referred to as 4E5A herein)        and/or antibody 4E11, e.g., at least 2, 3, 4, 5, 6, 8, 10, 12,        15, 100, 1,000, 5,000, or 10,000-fold greater affinity,    -   d) Neutralizes dengue virus (e.g., one or more of DV-1, DV-2,        DV-3, and DV-4, e.g., all of DV-1, DV-2, DV-3, and DV-4), e.g.,        in a focus reduction neutralization test or a related test for        evaluating neutralization of viral activity,    -   e) Neutralizes DV-4 with an improved IC50 compared to antibody        A11 and/or antibody 4E11, e.g., at least 2, 3, 4, 5, 6, 8, 10,        12, 25, 50, 75, 100, or 1,000-fold improved IC50, e.g., in a        focus reduction neutralization test or a related test for        evaluating neutralization of viral activity,    -   f) Has a mutation (e.g., one or more of a deletion, an        insertion, a substitution, e.g., a conservative substitution) at        one or more positions relative to A11, e.g., in the VH and/or        VL, e.g., in one or more CDRs or framework regions,    -   g) Has a mutation (e.g., one or more of a deletion, an        insertion, a substitution, e.g., a conservative substitution),        e.g., a substitution, e.g., a T33V substitution, in the heavy        chain CDR1 region relative to A11,    -   h) Has a mutation (e.g., one or more of a deletion, an        insertion, a substitution, e.g., a conservative substitution),        e.g., a deletion, at position 26 in the heavy chain FW1 relative        to A11,    -   i) Has both a mutation (e.g., one or more of a deletion, an        insertion, a substitution, e.g., a conservative substitution),        e.g., a substitution, e.g., a T33V mutation in the heavy chain        CDR1 region relative to A11 and a mutation (e.g., one or more of        a deletion, an insertion, a substitution, e.g., a conservative        substitution), e.g., a deletion, at position 26 in the heavy        chain FW1 relative to A11,    -   j) Has a mutation (e.g., one or more of a deletion, an        insertion, a substitution, e.g., a conservative substitution),        e.g., a substitution, e.g., a T33V mutation in the heavy chain        CDR1 region relative to A11, and has improved (e.g., relative to        A11) binding to and/or neutralization of dengue virus, e.g., to        one or more (e.g., all) of DV-1, DV-2, DV-3, and DV-4,    -   k) Has a mutation (e.g., one or more of a deletion, an        insertion, a substitution, e.g., a conservative substitution),        e.g., a deletion, at position 26 in the heavy chain FW1 relative        to A11, and has improved (e.g., relative to A11) binding to        and/or neutralization of dengue virus, e.g., to one or more        (e.g., all) of DV-1, DV-2, DV-3, and DV-4, e.g., to DV-4,    -   l) Has both a mutation (e.g., one or more of a deletion, an        insertion, a substitution, e.g., a conservative substitution),        e.g., a substitution, e.g., a T33V mutation in the heavy chain        CDR1 region relative to A11 and a mutation (e.g., one or more of        a deletion, an insertion, a substitution, e.g., a conservative        substitution), e.g., a deletion, at position 26 in the heavy        chain FW1 relative to A11, and has improved (e.g., relative to        A11) binding to and/or neutralization of dengue virus, e.g., to        one or more (e.g., all) of DV-1, DV-2, DV-3, and DV-4, e.g., to        DV-4,    -   m) Displays improved binding to EDIII of one or more (e.g., all)        of the dengue virus strains listed in FIGS. 10A-10B, 11 and        19-21, e.g., one or more DV-2 strains, one or more DV-3 strains,        one or more DV-4 strains, e.g., one or more of: DENV-4 BC2,        DENV-4-Sing10, DENV-4 NewCal09, DENV-4 Phil56, DENV-3 Sing09,        DENV-3 Nic10, DENV-3 H87, DENV-2 Peru95, DENV-2 Sing08, DENV-2        NGC, DENV-1 Hawaii/1944, DENV-2 New Guinea/1944 (NGC), DENV-3        Philippines/1956 (H87), DENV-4 Mexico/1997 (BC287/97), and        DENV-4 H241, e.g., with at least 2, 3, 4, 5, 6, 8, 10, 12, 25,        50, 75, 100, or 1,000-fold greater affinity,    -   n) Disrupts the native structure of the E protein on the surface        of the virion, e.g., which may cause inactivation of the virus,    -   o) Binds specifically to an epitope on EDIII, e.g., the same or        similar epitope as the epitope recognized by a A11 or B11        monoclonal antibody,    -   p) Shows the same or similar binding affinity or specificity, or        both, as an antibody of Table 1, e.g., D88, A48, F38, F108, or        C88,    -   q) Shows the same or similar binding affinity or specificity, or        both, as an antibody molecule (e.g., a heavy chain variable        region and light chain variable region) described in Table 1,        e.g., D88, A48, F38, F108, or C88,    -   r) Shows the same or similar binding affinity or specificity, or        both, as an antibody molecule (e.g., a heavy chain variable        region and light chain variable region) comprising an amino acid        sequence shown in Table 2,    -   s) Inhibits, e.g., competitively inhibits, the binding of a        second antibody molecule to EDIII wherein the second antibody        molecule is an antibody molecule described herein, e.g., an        antibody molecule chosen from Table 1, e.g., D88, A48, F38,        F108, or C88,    -   t) Binds the same or an overlapping epitope with a second        antibody molecule to EDIII, wherein the second antibody molecule        is an antibody molecule described herein, e.g., an antibody        molecule chosen from Table 1, e.g., D88, A48, F38, F108, or C88,    -   u) Competes for binding and binds the same epitope, with a        second antibody molecule to EDIII, wherein the second antibody        molecule is an antibody molecule described herein, e.g., an        antibody molecule chosen from Table 1, e.g., D88, A48, F38,        F108, or C88,    -   v) Has one or more biological properties of an antibody molecule        described herein, e.g., an antibody molecule chosen from Table        1, e.g., D88, A48, F38, F108, or C88,    -   w) Has one or more pharmacokinetic properties of an antibody        molecule described herein, e.g., an antibody molecule chosen        from Table 1, e.g., D88, A48, F38, F108, or C88, or    -   x) Inhibits one of more activities of dengue virus, e.g.,        neutralizes the virus (for instance, measured in a focus        reduction neutralization test or a related test for evaluating        neutralization of viral activity).

In some embodiments, the antibody molecule has a mutation (e.g., one ormore of a deletion, an insertion, a substitution, e.g., a conservativesubstitution), e.g., a T33V mutation in the heavy chain CDR 1 regionrelative to A11, in combination with one or more functional propertiesof List 1 above, e.g., one or more (e.g., two, three, four, five, six,seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen,sixteen, or all) of properties (a), (b), (c), (d), (e), (m), (n), (o),(p), (q), (r), (s), (t), (u), (v), (w), or (x). In certain embodiments,the antibody molecule has a mutation (e.g., one or more of a deletion,an insertion, a substitution, e.g., a conservative substitution), e.g.,a deletion, at position 26 in the heavy chain FW1 relative to A11, incombination with one or more functional properties of List 1 above,e.g., one or more (e.g., two, three, four, five, six, seven, eight,nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, or all)of properties (a), (b), (c), (d), (e), (m), (n), (o), (p), (q), (r),(s), (t), (u), (v), (w), or (x). In some embodiments, the antibodymolecule has both a mutation (e.g., one or more of a deletion, aninsertion, a substitution, e.g., a conservative substitution), e.g., aT33V mutation in the heavy chain CDR1 region relative to A11 and amutation (e.g., one or more of a deletion, an insertion, a substitution,e.g., a conservative substitution), e.g., a deletion, at position 26 inthe heavy chain FW1 relative to A11, in combination with one or morefunctional properties of List 1 above, e.g., one or more (e.g., two,three, four, five, six, seven, eight, nine, ten, eleven, twelve,thirteen, fourteen, fifteen, sixteen, or all) of properties (a), (b),(c), (d), (e), (m), (n), (o), (p), (q), (r), (s), (t), (u), (v), (w), or(x).

In some embodiments, the antibody molecule has, a T33V mutation in theheavy chain CDR 1 region relative to A11, in combination with one ormore functional properties of List 1 above, e.g., one or more (e.g.,two, three, four, five, six, seven, eight, nine, ten, eleven, twelve,thirteen, fourteen, fifteen, sixteen, or all) of properties (a), (b),(c), (d), (e), (m), (n), (o), (p), (q), (r), (s), (t), (u), (v), (w), or(x). In certain embodiments, the antibody molecule has a deletion atposition 26 in the heavy chain FW1 relative to A11, in combination withone or more functional properties of List 1 above, e.g., one or more(e.g., two, three, four, five, six, seven, eight, nine, ten, eleven,twelve, thirteen, fourteen, fifteen, sixteen, or all) of properties (a),(b), (c), (d), (e), (m), (n), (o), (p), (q), (r), (s), (t), (u), (v),(w), or (x). In some embodiments, the antibody molecule has both a T33Vmutation in the heavy chain CDR1 region relative to A11 and a deletionat position 26 in the heavy chain FW1 relative to A11, in combinationwith one or more functional properties of List 1 above, e.g., one ormore (e.g., two, three, four, five, six, seven, eight, nine, ten,eleven, twelve, thirteen, fourteen, fifteen, sixteen, or all) ofproperties (a), (b), (c), (d), (e), (m), (n), (o), (p), (q), (r), (s),(t), (u), (v), (w), or (x).

In certain embodiments, the antibody molecule has both a T33V mutationin the heavy chain CDR1 region relative to A11 and a deletion, atposition 26 in the heavy chain FW1 relative to A11, combination with oneor more functional properties of List 1 above, e.g., one or more (e.g.,two, three, four, five, six, seven, eight, nine, ten, eleven, twelve,thirteen, fourteen, fifteen, sixteen, or all) of properties (a), (b),(c), (d), (e), (m), (n), (o), (p), (q), (r), (s), (t), (u), (v), (w),(x). For example, the antibody molecule may bind EDIII (e.g., of DV-1,DV-2, DV-3, or DV-4) with high affinity, e.g., with a dissociationconstant (K_(D)) of less than about 100 nM, typically about 10 nM, andmore typically, about 10-0.01 nM, about 5-0.01 nM, about 3-0.05 nM, orabout 1-0.1 nM, or stronger, e.g., less than about 80, 70, 60, 50, 40,30, 20, 10, 8, 6, 4, 3, 2, 1, 0.5, 0.2, 0.1, 0.05, or 0.01 nM. As afurther example, the antibody molecule may bind to DV-4 EDIII with highaffinity, e.g., with a dissociation constant (K_(D)) of less than about100 nM, e.g., about 10 nM, e.g., about 10-1 nM or stronger, e.g., lessthan about 10, 8, 6, 5, 4, or 3 nM. Furthermore, the antibody moleculemay neutralize DV-4 with an improved IC50 compared to antibody A11and/or antibody 4E11, e.g., at least 2, 3, 4, 5, 6, 8, 10, 12, 100,1,000-fold improved IC50, e.g., in a focus reduction neutralization testor a related test for evaluating neutralization of viral activity.

In certain embodiments, affinity is measured by competition ELISA orSPR. In some embodiments, affinity is measured by one or more ofBlAcore, ELISA, or flow cytometry.

In certain embodiments, the anti-dengue antibody molecule is a humanizedantibody molecule and has one or more properties from List 1 above,e.g., one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, or all) of properties (a), (b), (c), (d), (e), (m), (n), (o),(p), (q), (r), (s), (t), (u), (v), (w), or (x).

In some embodiments, the antibody molecule binds to EDIII with highaffinity, e.g., with a K_(D) that is at least about 10%, 20%, 30%, 40%,50%, 60%, 70%, 80% or 90% lower than the K_(D) of a murine anti-dengueantibody molecule, e.g., 4E11, A11 or B11.

In some embodiments, the expression level of the antibody molecule ishigher, e.g., at least about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10-foldhigher, than the expression level of a murine antibody molecule, e.g., amurine anti-dengue antibody molecule described herein. In someembodiments, the antibody molecule is expressed in mammalian cells,e.g., human or rodent cells.

In some embodiments, the antibody molecule reduces one or more denguevirus activities with an IC50 (concentration at 50% inhibition) that islower, e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or90% lower, than the IC50 of a murine anti-dengue antibody molecule,e.g., a murine anti-dengue antibody molecule described herein. In someembodiments, the dengue virus activity is neutralized, e.g., in thefocus reduction neutralization test described in Tharakaramana et al.,Proc Natl Acad Sci USA. 2013 Apr. 23; 110(17):E1555-64. doi:10.1073/pnas.1303645110. Epub 2013 Apr. 8, which is hereby expresslyincorporated by reference in its entirety, including all supplementalmaterials. Other related tests that can be used to evaluateneutralization of viral activity include, e.g., enzyme-linkedimmunosorbent assay (ELISA)-based microneutralization (MN) assays (e.g.,as described in Vorndam et al., Am J Trop Med Hyg 2002; 66: 208-212) andfluorescent antibody cell sorter-based, DC-SIGN expresser dendritic cell(DC) assay (e.g., as described in Martin et al., J Virol Methods 2006;134: 74-85).

In certain embodiments, the antibody molecule reduces transmission ofdengue virus (e.g., reduces transmission between a subject (e.g., ahuman) and a mosquito), e.g., by at least about 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, or 90%, e.g., as measured by a mosquito model describedherein.

In other embodiments, the antibody molecule has an improved ability toreduce transmission of dengue virus (e.g., has an improved ability toreduce transmission of dengue virus between a subject (e.g., a human)and a mosquito), e.g., by at least about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9or 10-fold higher than a murine anti-dengue antibody molecule, e.g., amurine anti-dengue antibody molecule described herein, e.g., 4E11, A11or B11, e.g., as measured by a mosquito model described herein.

In other embodiments, the antibody molecule reduces the mosquito viralload (e.g., the amount of virus, and/or infectivity, carried by amosquito), e.g., by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, or 90%, e.g., as measured by a mosquito model described herein.

In some embodiments, the antibody molecule has improved stability, e.g.,at least about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10-fold more stable invivo or in vitro, than a murine anti-dengue antibody molecule, e.g., amurine anti-dengue antibody molecule described herein, e.g., 4E11, A11or B11.

In some embodiments, the anti-dengue antibody molecule comprises atleast one antigen-binding region, e.g., a variable region or anantigen-binding fragment thereof, from an antibody described herein,e.g., an antibody chosen from Table 1, e.g., D88, A48, F38, F108, orC88, or a sequence substantially identical to any of the aforesaidsequences (e.g., a sequence at least about 85%, 90%, 95%, 99% or moreidentical thereto, and/or having one, two, three or more substitutions,insertions or deletions, e.g., conserved substitutions). In someembodiments, an antibody molecule has a structural feature discussed inthis paragraph and one or more advantageous properties such as animproved (e.g., relative to A11) affinity for or neutralization activitytowards dengue virus, e.g., DV-4. In some embodiments, the advantageousproperty is a property of List 1, e.g., one or more (e.g., two, three,four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen,fourteen, fifteen, sixteen, or all) of properties (a), (b), (c), (d),(e), (m), (n), (o), (p), (q), (r), (s), (t), (u), (v), (w), or (x).

In certain embodiments, the anti-dengue antibody molecule comprises atleast one, two, three, or four variable regions from an antibodydescribed herein, e.g., an antibody of Table 1, e.g., D88, A48, F38,F108, or C88, or a sequence substantially identical to any of theaforesaid sequences (e.g., a sequence at least about 85%, 90%, 95%, 99%or more identical thereto, and/or having one, two, three or moresubstitutions, insertions or deletions, e.g., conserved substitutions).In some embodiments, an antibody molecule has a structural featurediscussed in this paragraph and one or more advantageous properties suchas an improved (e.g., relative to A11) affinity for or neutralizationactivity towards dengue virus, e.g., DV-4. In some embodiments, theadvantageous property is a property of List 1, e.g., one or more (e.g.,two, three, four, five, six, seven, eight, nine, ten, eleven, twelve,thirteen, fourteen, fifteen, sixteen, or all) of properties (a), (b),(c), (d), (e), (m), (n), (o), (p), (q), (r), (s), (t), (u), (v), (w), or(x).

In some embodiments, the anti-dengue antibody molecule comprises atleast one or two heavy chain variable regions from an antibody describedherein, e.g., an antibody of Table 1, e.g., D88, A48, F38, F108, or C88,or a sequence substantially identical to any of the aforesaid sequences(e.g., a sequence at least about 85%, 90%, 95%, 99% or more identicalthereto, and/or having one, two, three or more substitutions, insertionsor deletions, e.g., conserved substitutions). In some embodiments, anantibody molecule has a structural feature discussed in this paragraphand one or more advantageous properties such as an improved (e.g.,relative to A11) affinity for or neutralization activity towards denguevirus, e.g., DV-4. In some embodiments, the advantageous property is aproperty of List 1, e.g., one or more (e.g., two, three, four, five,six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen,fifteen, sixteen, or all) of properties (a), (b), (c), (d), (e), (m),(n), (o), (p), (q), (r), (s), (t), (u), (v), (w), or (x).

In certain embodiments, the anti-dengue antibody molecule comprises atleast one or two light chain variable regions from an antibody describedherein, e.g., an antibody of Table 1, e.g., D88, A48, F38, F108, or C88or a sequence substantially identical to any of the aforesaid sequences(e.g., a sequence at least about 85%, 90%, 95%, 99% or more identicalthereto, and/or having one, two, three or more substitutions, insertionsor deletions, e.g., conserved substitutions). In some embodiments, anantibody molecule has a structural feature discussed in this paragraphand one or more advantageous properties such as an improved (e.g.,relative to A11) affinity for or neutralization activity towards denguevirus, e.g., DV-4. In some embodiments, the advantageous property is aproperty of List 1, e.g., one or more (e.g., two, three, four, five,six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen,fifteen, sixteen, or all) of properties (a), (b), (c), (d), (e), (m),(n), (o), (p), (q), (r), (s), (t), (u), (v), (w), or (x).

In certain embodiments, the anti-dengue antibody molecule comprises avaline at position 33 (e.g., a T33V mutation) in the VH region, e.g.,relative to 4E5A and/or 4E11 or an antibody of Table 1. In someembodiments, the anti-dengue antibody comprises a del26 (deletion atposition 26) in the VH region, e.g., relative to 4E5A or an antibody ofTable 1. In some embodiments, the anti-dengue antibody comprises both avaline at position 33 (e.g., a T33V mutation) and a del26 mutation inthe VH region, e.g., relative to 4E5A or an antibody of Table 1. In someembodiments, an antibody molecule has a structural feature discussed inthis paragraph and one or more advantageous properties such as animproved (e.g., relative to A11) affinity for or neutralization activitytowards dengue virus, e.g., DV-4. In some embodiments, the advantageousproperty is a property of List 1, e.g., one or more (e.g., two, three,four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen,fourteen, fifteen, sixteen, or all) of properties (a), (b), (c), (d),(e), (m), (n), (o), (p), (q), (r), (s), (t), (u), (v), (w), or (x).

In some embodiments, the anti-dengue antibody molecule comprises atleast one, two, or three complementarity determining regions (CDRs) froma heavy chain variable region of an antibody described herein, e.g., anantibody of Table 1, e.g., D88, A48, F38, F108, or C88, or a sequencesubstantially identical to any of the aforesaid sequences (e.g., asequence at least about 85%, 90%, 95%, 99% or more identical thereto,and/or having one, two, three or more substitutions, insertions ordeletions, e.g., conserved substitutions). In some embodiments, anantibody molecule has a structural feature discussed in this paragraphand one or more advantageous properties such as an improved (e.g.,relative to A11) affinity for or neutralization activity towards denguevirus, e.g., DV-4. In some embodiments, the advantageous property is aproperty of List 1, e.g., one or more (e.g., two, three, four, five,six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen,fifteen, sixteen, or all) of properties (a), (b), (c), (d), (e), (m),(n), (o), (p), (q), (r), (s), (t), (u), (v), (w), or (x).

In certain embodiments, the anti-dengue antibody molecule includes atleast one, two, or three CDRs (or collectively all of the CDRs) from aheavy chain variable region comprising an amino acid sequence shown inTable 3. In some embodiments, one or more of the CDRs (or collectivelyall of the CDRs) have one, two, three, four, five, six or more changes,e.g., amino acid substitutions, insertions, or deletions, relative tothe CDRs shown in Table 3. In some embodiments, an antibody molecule hasa structural feature discussed in this paragraph and one or moreadvantageous properties such as an improved (e.g., relative to A11)affinity for or neutralization activity towards dengue virus, e.g.,DV-4. In some embodiments, the advantageous property is a property ofList 1, e.g., one or more (e.g., two, three, four, five, six, seven,eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen,or all) of properties (a), (b), (c), (d), (e), (m), (n), (o), (p), (q),(r), (s), (t), (u), (v), (w), or (x).

In some embodiments, the anti-dengue antibody molecule includes at leastone, two, or three CDRs (or collectively all of the CDRs) from a lightchain variable region comprising an amino acid sequence shown in Table3. In some embodiments, one or more of the CDRs (or collectively all ofthe CDRs) have one, two, three, four, five, six or more changes, e.g.,amino acid substitutions, insertions, or deletions, relative to the CDRsshown in Table 3. In some embodiments, an antibody molecule has astructural feature discussed in this paragraph and one or moreadvantageous properties such as an improved (e.g., relative to A11)affinity for or neutralization activity towards dengue virus, e.g.,DV-4. In some embodiments, the advantageous property is a property ofList 1, e.g., one or more (e.g., two, three, four, five, six, seven,eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen,or all) of properties. (a), (b), (c), (d), (e), (m), (n), (o), (p), (q),(r), (s), (t), (u), (v), (w), or (x).

In some embodiments, the anti-dengue antibody molecule includes at leastone, two, three, four, five or six CDRs (or collectively all of theCDRs) from a heavy and light chain variable region comprising an aminoacid sequence shown in Table 3. In some embodiments, one or more of theCDRs (or collectively all of the CDRs) have one, two, three, four, five,six or more changes, e.g., amino acid substitutions, insertions, ordeletions, relative to the CDRs shown in Table 3. In some embodiments,an antibody molecule has a structural feature discussed in thisparagraph and one or more advantageous properties such as an improved(e.g., relative to A11) affinity for or neutralization activity towardsdengue virus, e.g., DV-4. In some embodiments, the advantageous propertyis a property of List 1, e.g., one or more (e.g., two, three, four,five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen,fifteen, sixteen, or all) of properties (a), (b), (c), (d), (e), (m),(n), (o), (p), (q), (r), (s), (t), (u), (v), (w), or (x).

In certain embodiments, the anti-dengue antibody molecule includes allsix CDRs from an antibody described herein, e.g., an antibody of Table1, e.g., D88, A48, F38, F108, or C88, or closely related CDRs, e.g.,CDRs which are identical or which have at least one amino acidalteration, but not more than two, three or four alterations (e.g.,substitutions e.g., conservative substitutions, deletions, orinsertions). In certain embodiments, the anti-dengue antibody moleculemay include any CDR described herein. In some embodiments, an antibodymolecule has a structural feature discussed in this paragraph and one ormore advantageous properties such as an improved (e.g., relative to A11)affinity for or neutralization activity towards dengue virus, e.g.,DV-4. In some embodiments, the advantageous property is a property ofList 1, e.g., one or more (e.g., two, three, four, five, six, seven,eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen,or all) of properties (a), (b), (c), (d), (e), (m), (n), (o), (p), (q),(r), (s), (t), (u), (v), (w), or (x).

In some embodiments, the anti-dengue antibody molecule includes at leastone, two, or three Chothia hypervariable loops from a heavy chainvariable region of an antibody described herein, e.g., an antibody ofTable 1, e.g., D88, A48, F38, F108, or C88, or at least the amino acidsfrom those hypervariable loops that contact EDIII. For instance, in someembodiments, an antibody molecule provided herein has a VHCDR1 of SEQ IDNO: 9, a VHCDR2 of SEQ ID NO: 10, and a VHCDR3 of SEQ ID NO: 5. Anantibody molecule provided herein may also have a VHCDR1 of SEQ ID NO:15, a VHCDR2 of SEQ ID NO: 10, and a VHCDR3 of SEQ ID NO: 5. An antibodymolecule provided herein may also have a VHCDR1 of SEQ ID NO: 22, 24,26, 28, or 30; a VHCDR2 of SEQ ID NO: 10; and a VHCDR3 of SEQ ID NO: 5.In some embodiments, an antibody molecule has a structural featurediscussed in this paragraph and one or more advantageous properties suchas an improved (e.g., relative to A11) affinity for or neutralizationactivity towards dengue virus, e.g., DV-4. In some embodiments, theadvantageous property is a property of List 1, e.g., one or more (e.g.,two, three, four, five, six, seven, eight, nine, ten, eleven, twelve,thirteen, fourteen, fifteen, sixteen, or all) of properties (a), (b),(c), (d), (e), (m), (n), (o), (p), (q), (r), (s), (t), (u), (v), (w), or(x).

In some embodiments, and optionally in combination with heavy chain CDRsdescribed herein, the anti-dengue antibody molecule includes at leastone, two, or three Chothia hypervariable loops from a light chainvariable region of an antibody described herein, e.g., an antibody ofTable 1, e.g., D88, A48, F38, F108, or C88, or at least the amino acidsfrom those hypervariable loops that contact EDIII. For instance, incertain embodiments, an antibody molecule provided herein has a VHCDR1of SEQ ID NO: 6, a VHCDR2 of SEQ ID NO: 7, and a VHCDR3 of SEQ ID NO: 8.In some embodiments, an antibody molecule has a structural featurediscussed in this paragraph and one or more advantageous properties suchas an improved (e.g., relative to A11) affinity for or neutralizationactivity towards dengue virus, e.g., DV-4. In some embodiments, theadvantageous property is a property of List 1, e.g., one or more (e.g.,two, three, four, five, six, seven, eight, nine, ten, eleven, twelve,thirteen, fourteen, fifteen, sixteen, or all) of properties (a), (b),(c), (d), (e), (m), (n), (o), (p), (q), (r), (s), (t), (u), (v), (w), or(x).

In some embodiments, the anti-dengue antibody molecule includes at leastone, two, or three Kabat hypervariable loops from a heavy chain variableregion of an antibody described herein, e.g., an antibody of Table 1,e.g., D88, A48, F38, F108, or C88, or at least the amino acids fromthose hypervariable loops that contact EDIII. In some embodiments, anantibody molecule has a structural feature discussed in this paragraphand one or more advantageous properties such as an improved (e.g.,relative to A11) affinity for or neutralization activity towards denguevirus, e.g., DV-4. In some embodiments, the advantageous property is aproperty of List 1, e.g., one or more (e.g., two, three, four, five,six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen,fifteen, sixteen, or all) of properties (a), (b), (c), (d), (e), (m),(n), (o), (p), (q), (r), (s), (t), (u), (v), (w), or (x).

In some embodiments, the anti-dengue antibody molecule includes at leastone, two, or three Kabat hypervariable loops from a light chain variableregion of an antibody described herein, e.g., an antibody of Table 1,e.g., D88, A48, F38, F108, or C88, or at least the amino acids fromthose hypervariable loops that contact EDIII. In some embodiments, anantibody molecule has a structural feature discussed in this paragraphand one or more advantageous properties such as an improved (e.g.,relative to A11) affinity for or neutralization activity towards denguevirus, e.g., DV-4. In some embodiments, the advantageous property is aproperty of List 1, e.g., one or more (e.g., two, three, four, five,six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen,fifteen, sixteen, or all) of properties (a), (b), (c), (d), (e), (m),(n), (o), (p), (q), (r), (s), (t), (u), (v), (w), or (x).

In certain embodiments, the anti-dengue antibody molecule includes atleast one, two, three, four, five, or six hypervariable loops from theheavy and light chain variable regions of an antibody described herein,e.g., an antibody of Table 1, e.g., D88, A48, F38, F108, or C88, or atleast the amino acids from those hypervariable loops that contact EDIII.In some embodiments, an antibody molecule has a structural featurediscussed in this paragraph and one or more advantageous properties suchas an improved (e.g., relative to A11) affinity for or neutralizationactivity towards dengue virus, e.g., DV-4. In some embodiments, theadvantageous property is a property of List 1, e.g., one or more (e.g.,two, three, four, five, six, seven, eight, nine, ten, eleven, twelve,thirteen, fourteen, fifteen, sixteen, or all) of properties (a), (b),(c), (d), (e), (m), (n), (o), (p), (q), (r), (s), (t), (u), (v), (w), or(x).

In certain embodiments, the anti-dengue antibody molecule includes allsix hypervariable loops from the heavy and light chain variable regionsof an antibody described herein, e.g., an antibody of Table 1, e.g.,D88, A48, F38, F108, or C88, or at least the amino acids from thosehypervariable loops that contact EDIII, or closely related hypervariableloops, e.g., hypervariable loops which are identical or which have atleast one amino acid alteration, but not more than two, three or fouralterations (e.g., substitutions, e.g., conservative substitutions,deletions, or insertions). In some embodiments, an antibody molecule hasa structural feature discussed in this paragraph and one or moreadvantageous properties such as an improved (e.g., relative to A11)affinity for or neutralization activity towards dengue virus, e.g.,DV-4. In some embodiments, the advantageous property is a property ofList 1, e.g., one or more (e.g., two, three, four, five, six, seven,eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen,or all) of properties (a), (b), (c), (d), (e), (m), (n), (o), (p), (q),(r), (s), (t), (u), (v), (w), or (x).

In some embodiments, the anti-dengue antibody molecule includes at leastone, two, or three hypervariable loops that have the same canonicalstructures as the corresponding hypervariable loop of an antibodydescribed herein, e.g., an antibody of Table 1, e.g., D88, A48, F38,F108, or C88, e.g., the same canonical structures as at least loop 1and/or loop 2 of the heavy and/or light chain variable domains of anantibody described herein. See, e.g., Chothia et al., (1992) J. Mol.Biol. 227:799-817; Tomlinson et al., (1992) J. Mol. Biol. 227:776-798for descriptions of hypervariable loop canonical structures. Thesestructures can be determined by inspection of the tables described inthese publications. In some embodiments, an antibody molecule has astructural feature discussed in this paragraph and one or moreadvantageous properties such as an improved (e.g., relative to A11)affinity for or neutralization activity towards dengue virus, e.g.,DV-4. In some embodiments, the advantageous property is a property ofList 1, e.g., one or more (e.g., two, three, four, five, six, seven,eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen,or all) of properties (a), (b), (c), (d), (e), (m), (n), (o), (p), (q),(r), (s), (t), (u), (v), (w), or (x).

In certain embodiments, the anti-dengue antibody molecule comprises atleast one, two, or three (e.g., all) CDRs from a heavy chain variableregion having an amino acid sequence as set forth in Table 3, or asequence substantially identical thereto (e.g., a sequence at leastabout 85%, 90%, 95%, 99% or more identical thereto, and/or having one,two, three or more substitutions, insertions or deletions, e.g.,conserved substitutions). In some embodiments, the anti-dengue antibodymolecule comprises at least one, two, or three (e.g., all) CDRs from alight chain variable region having an amino acid sequence as set forthin Table 3, or a sequence substantially identical thereto (e.g., asequence at least about 85%, 90%, 95%, 99% or more identical thereto,and/or having one, two, three or more substitutions, insertions ordeletions, e.g., conserved substitutions). In certain embodiments, theanti-dengue antibody molecule comprises at least one, two, three, four,five or six (e.g., all) CDRs from heavy and light chain variable regionshaving an amino acid sequence as set forth in Table 3, or a sequencesubstantially identical thereto (e.g., a sequence at least about 85%,90%, 95%, 99% or more identical thereto, and/or having one, two, threeor more substitutions, insertions or deletions, e.g., conservedsubstitutions). In some embodiments, an antibody molecule has astructural feature discussed in this paragraph and one or moreadvantageous properties such as an improved affinity for orneutralization activity towards dengue virus, e.g., DV-4. In someembodiments, the advantageous property is a property of List 1, e.g.,one or more (e.g., two, three, four, five, six, seven, eight, nine, ten,eleven, twelve, thirteen, fourteen, fifteen, sixteen, or all) ofproperties (a), (b), (c), (d), (e), (m), (n), (o), (p), (q), (r), (s),(t), (u), (v), (w), or (x).

In some embodiments, the anti-dengue antibody molecule comprises atleast one, two, or three (e.g., all) CDRs from a heavy chain variableregion having an amino acid sequence of an antibody described herein,e.g., an antibody of Table 1, e.g., D88, A48, F38, F108, or C88, or asequence substantially identical thereto (e.g., a sequence at leastabout 85%, 90%, 95%, 99% or more identical thereto, and/or having one,two, three or more substitutions, insertions or deletions, e.g.,conserved substitutions). In certain embodiments, the anti-dengueantibody molecule comprises at least one, two, or three (e.g., all) CDRsfrom a light chain variable region having an amino acid sequence of anantibody described herein, e.g., an antibody of Table 1, e.g., D88, A48,F38, F108, or C88, or a sequence substantially identical thereto (e.g.,a sequence at least about 85%, 90%, 95%, 99% or more identical thereto,and/or having one, two, three or more substitutions, insertions ordeletions, e.g., conserved substitutions). In some embodiments, theanti-dengue antibody molecule comprises six CDRs described herein, e.g.,in a VL and VH sequence of Table 2. In some embodiments, an antibodymolecule has a structural feature discussed in this paragraph and one ormore advantageous properties such as an improved (e.g., relative to A11)affinity for or neutralization activity towards dengue virus, e.g.,DV-4. In some embodiments, the advantageous property is a property ofList 1, e.g., one or more (e.g., two, three, four, five, six, seven,eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen,or all) of properties (a), (b), (c), (d), (e), (m), (n), (o), (p), (q),(r), (s), (t), (u), (v), (w), or (x).

In aspects embodiments, the antibody molecule has a VHCDR1, VHCDR2,VHCDR3, VLCDR1, VLCDR2, and VLCDR3 selected from Table 3. The six CDRsmay all be Kabat-defined, all Chothia-defined, or some Kabat- and someChothia-defined. For instance, VHCDR1 may be selected from SEQ ID NO: 3,9, 14, 15, 22, 24, 26, 28, or 30; VHCDR2 may be selected from SE$Q IDNO: 4, 10, or 35; VHCDR3 may be SEQ ID NO: 5; VLCDR1 may be SEQ ID NO:6; VLCDR2 may be SEQ ID NO: 7; and VLCDR3 may be SEQ ID NO: 8.

In certain embodiments, the light or the heavy chain variable frameworkof the anti-dengue antibody can be chosen from: (a) a light or heavychain variable framework including at least 80%, 85%, 87% 90%, 92%, 93%,95%, 97%, 98%, or preferably 100% of the amino acid residues from ahuman light or heavy chain variable framework, e.g., a light or heavychain variable framework residue from a human mature antibody, a humangermline sequence, or a human consensus sequence; (b) a light or heavychain variable framework including from 20% to 80%, 40% to 60%, 60% to90%, or 70% to 95% of the amino acid residues from a human light orheavy chain variable framework, e.g., a light or heavy chain variableframework residue from a human mature antibody, a human germlinesequence, or a human consensus sequence; (c) a non-human framework(e.g., a rodent framework); or (d) a non-human framework that has beenmodified, e.g., to remove antigenic or cytotoxic determinants, e.g.,deimmunized, or partially humanized. In some embodiments, the light orheavy chain variable framework region includes a light or heavy chainvariable framework sequence at least 70, 75, 80, 85, 87, 88, 90, 92, 94,95, 96, 97, 98, 99% identical or identical to the frameworks of a VH orVL segment of a human germline gene. In some embodiments, an antibodymolecule has a structural feature discussed in this paragraph and one ormore advantageous properties such as an improved (e.g., relative to A11)affinity for or neutralization activity towards dengue virus, e.g.,DV-4. In some embodiments, the advantageous property is a property ofList 1, e.g., one or more (e.g., two, three, four, five, six, seven,eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen,or all) of properties (a), (b), (c), (d), (e), (m), (n), (o), (p), (q),(r), (s), (t), (u), (v), (w), or (x).

In certain, embodiments, the VH region (e.g., the framework regionstherein) of the anti-dengue antibody comprises one or more positionsfrom a human VH region, e.g., human heavy chain germline-encoded aminoacid sequences, e.g., positions found in one or more (e.g., all) of FW1,FW2, FW3, and FW4. In certain embodiments, optionally in combinationwith the VH residues discussed in the previous sentence, the VL region(e.g., the framework regions therein) of the anti-dengue antibodycomprises one or more positions from a human VL region, e.g., humanheavy chain germline-encoded amino acid sequences, e.g., positions foundin one or more (e.g., two, three, four, five, or all) of FW1, FW2, FW3,and FW4.

For example, in some embodiments, the antibody molecule comprises one ormore (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, or all)residues according to heavy chain or light chain FW1, FW2, FW3, or FW4regions from a human germline sequence of Table 5. More specifically, insome embodiments the antibody molecule has one or more (e.g., at least2, 3, 4, 5, 10, or 15, or all) VH FW1 residues of a VH germline sequenceof Table 6; in some embodiments the antibody molecule has one or more(e.g., at least 2, 3, 4, 5, 10, or 15, or all) VH FW2 residues of a VHgermline sequence of Table 6; in some embodiments the antibody moleculehas one or more (e.g., at least 2, 3, 4, 5, 10, or 15, or all) VH FW3residues of a VH germline sequence of Table 6, and in some embodimentsthe antibody molecule has one or more (e.g., at least 2, 3, 4, 5, 10, or15, or all) VH FW4 residues of a VH germline sequence of Table 6.Furthermore, and optionally in combination with the heavy chain residuesdiscussed in the previous sentence, in some embodiments the antibodymolecule has one or more (e.g., at least 2, 3, 4, 5, 10, or 15, or all)VL FW1 residues of a VL germline sequence of Table 6; in someembodiments the antibody molecule has one or more (e.g., at least 2, 3,4, 5, 10, or 15, or all) VL FW2 residues of a VL germline sequence ofTable 6; in some embodiments the antibody molecule has one or more(e.g., at least 2, 3, 4, 5, 10, or 15, or all) VL FW3 residues of a VLgermline sequence of Table 6, and in some embodiments the antibodymolecule has one or more (e.g., at least 2, 3, 4, 5, 10, or 15, or all)VL FW4 residues of a VL germline sequence of Table 6. In certainembodiments, the antibody molecule has a heavy chain frameworkVH1-18*01, JH4*01 and/or light chain framework Vk4-1*01, Jk2*02.

In certain embodiments, the anti-dengue antibody molecule comprises aheavy chain variable domain having at least one, two, three, four, five,six, seven, ten, fifteen, twenty or more changes, e.g., amino acidsubstitutions, insertions, or deletions, from an amino acid sequence ofTable 1, e.g., B11, D88, A48, F38, F108, or C88, e.g., the amino acidsequence of the FR region in the entire variable region. In someembodiments, the anti-dengue antibody molecule comprises a heavy chainvariable domain having one or more (e.g., at least 5, 10, 15, or 20, orall) of: Q at position 3, V at position 5, a deletion of E at position6, V at position 12, K at position 13, K at position 20, V at position21, K at position 24, a deletion of S at position 26, V at position 33,R at position 39, A at position 41, G at position 43, M at position 49,L at position 65, Rat position 68, V at position 69, M at position 71, Tat position 77, M at position 82, E at position 83, R at position 85, Rat position 88, D at position 90, A or V or S at position 98, and S atposition 117 of the amino acid sequence of an antibody of Table 1, e.g.,A11. Examples of antibodies having one or more (e.g., all) of thesemutations include A48, B48, C88, F38, F108, and D48. In someembodiments, the humanized heavy chain contains one or more of: Q atposition 3, V at position 5, a deletion of E at position 6, V atposition 12, K at position 13, K at position 20, V at position 21, K atposition 24, R at position 39, A at position 41, G at position 43, M atposition 49, L at position 65, R at position 68, V at position 69, M atposition 71, Tat position 77, M at position 82, E at position 83, R atposition 85, R at position 88, D at position 90, V or A or S at position98, and S at position 117 of the amino acid sequence of an antibody ofTable 1, e.g., A11. An example of an antibody having one or more (e.g.,all) of these mutations is A68. In some embodiments, an antibodymolecule has a structural feature discussed in this paragraph and one ormore advantageous properties such as an improved (e.g., relative to A11)affinity for or neutralization activity towards dengue virus, e.g.,DV-4. In some embodiments, the advantageous property is a property ofList 1, e.g., one or more (e.g., two, three, four, five, six, seven,eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen,or all) of properties (a), (b), (c), (d), (e), (m), (n), (o), (p), (q),(r), (s), (t), (u), (v), (w), or (x).

In certain embodiments (and optionally in combination with the heavychain substitutions described herein, e.g., in the previous paragraph),the anti-dengue antibody molecule comprises a light chain variabledomain having at least one, two, three, four, five, six, seven, ten,fifteen, twenty or more amino acid changes, e.g., amino acidsubstitutions, insertions, or deletions, from an amino acid sequence ofTable 1, e.g., B11, D88, A48, F38, F108, or C88, e.g., the amino acidsequence of the FR region in the entire variable region. In certainembodiments, the anti-dengue antibody comprises a light chain variabledomain having one or more (e.g., at least 5, 10, 15, or all) of: D atposition 1, I at position 2, S at position 7, E at position 17, P atposition 44, V at position 62, D at position 64, G at position 72, S atposition 80, S at position 81, L at position 82, Q at position 83, E atposition 85, V at position 89, Y at position 91, and Q at position 104of the amino acid sequence of an antibody of Table 1, e.g., B11, D88,A48, F38, F108, or C88. In some embodiments, an antibody molecule has astructural feature discussed in this paragraph and one or moreadvantageous properties such as an improved (e.g., relative to A11)affinity for or neutralization activity towards dengue virus, e.g.,DV-4. In some embodiments, the advantageous property is a property ofList 1, e.g., one or more (e.g., two, three, four, five, six, seven,eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen,or all) of properties (a), (b), (c), (d), (e), (m), (n), (o), (p), (q),(r), (s), (t), (u), (v), (w), or (x).

In some embodiments, the heavy or light chain variable domain, or both,of the of the anti-dengue antibody molecule includes an amino acidsequence, which is substantially identical to an amino acid disclosedherein, e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higheridentical to a variable region of an antibody described herein, e.g., anantibody of Table 1, e.g., D88, A48, F38, F108, or C88, or which differsby at least 1, 2, 3, 4, or 5 residues, but less than 40, 30, 20, or 10residues, from a variable region of an antibody described herein. Insome embodiments, an antibody molecule has a structural featurediscussed in this paragraph and one or more advantageous properties suchas an improved (e.g., relative to A11) affinity for or neutralizationactivity towards dengue virus, e.g., DV-4. In some embodiments, theadvantageous property is a property of List 1, e.g., one or more (e.g.,two, three, four, five, six, seven, eight, nine, ten, eleven, twelve,thirteen, fourteen, fifteen, sixteen, or all) of properties (a), (b),(c), (d), (e), (m), (n), (o), (p), (q), (r), (s), (t), (u), (v), (w), or(x).

In certain embodiments, the heavy or light chain variable region, orboth, of the anti-dengue antibody molecule includes an amino acidsequence encoded by a nucleic acid sequence described herein, or anucleic acid that hybridizes to a nucleic acid sequence that encodes anantibody of Table 1, e.g., D88, A48, F38, F108, or C88, or itscomplement, e.g., under low stringency, medium stringency, or highstringency, or other hybridization condition described herein. Thisapplication also discloses the heavy or light chain variable region, orboth, of the anti-dengue antibody molecule includes an amino acidsequence encoded by a nucleic acid sequence of Table 4, or itscomplement, e.g., under low stringency, medium stringency, or highstringency, or other hybridization condition described herein. In someembodiments, the nucleic acid is at least 80%, 85%, 90%, 92%, 95%, 97%,98%, 99% or higher identical to a sequence of Table 4 or a portionthereof. In some embodiments, an antibody molecule has a structuralfeature discussed in this paragraph and one or more advantageousproperties such as an improved (e.g., relative to A11) affinity for orneutralization activity towards dengue virus, e.g., DV-4. In someembodiments, the advantageous property is a property of List 1, e.g.,one or more (e.g., two, three, four, five, six, seven, eight, nine, ten,eleven, twelve, thirteen, fourteen, fifteen, sixteen, or all) ofproperties (a), (b), (c), (d), (e), (m), (n), (o), (p), (q), (r), (s),(t), (u), (v), (w), or (x).

In certain embodiments, the anti-dengue antibody molecule comprises atleast one, two, three, or four antigen-binding regions, e.g., variableregions, having an amino acid sequence as set forth in Table 2, or asequence substantially identical thereto (e.g., a sequence at leastabout 85%, 90%, 95%, 99% or more identical thereto, or which differs byno more than 1, 2, 5, 10, or 15 amino acid residues from the sequencesshown in Table 2). In certain embodiments, the anti-dengue antibodymolecule includes a VH and/or VL domain encoded by a nucleic acid havinga nucleotide sequence that encodes an antibody of Table 1, e.g., D88,A48, F38, F108, or C88, or a sequence substantially identical to any oneof the nucleotide sequences (e.g., a sequence at least about 85%, 90%,95%, 99% or more identical thereto, or which differs by no more than 3,6, 15, 30, or 45 nucleotides from any one of the nucleotide sequences).In some embodiments, an antibody molecule has a structural featurediscussed in this paragraph and one or more advantageous properties suchas an improved (e.g., relative to A11) affinity for or neutralizationactivity towards dengue virus, e.g., DV-4. In some embodiments, theadvantageous property is a property of List 1, e.g., one or more (e.g.,two, three, four, five, six, seven, eight, nine, ten, eleven, twelve,thirteen, fourteen, fifteen, sixteen, or all) of properties (a), (b),(c), (d), (e), (m), (n), (o), (p), (q), (r), (s), (t), (u), (v), (w), or(x).

In certain aspects, the present disclosure provides an antibodymolecule, optionally capable of binding dengue virus, comprising:

(a) a heavy chain immunoglobulin variable region segment comprising:

a CDR1 comprising the sequence DVYMS (SEQ ID NO: 3) (or a sequence thatdiffers by no more than, 1, 2, or 3 amino acids therefrom, optionallyprovided that V is unchanged),

a CDR2 comprising the sequence RIDPENGDTKYDPKLQG (SEQ ID NO: 4) (or asequence that differs by no more than, 1, 2, 3, 4, or 5 amino acidstherefrom, optionally provided that L is unchanged), and

a CDR3 comprising the sequence GWEGFAY (SEQ ID NO: 5) (or a sequencethat differs by no more than, 1, 2, or 3 amino acids therefrom);

(b) a light chain variable region segment comprising:

a CDR1 comprising the sequence RASENVDKYGNSFMH (SEQ ID NO: 6) (or asequence that differs by no more than, 1, 2, 3, 4, or 5 amino acidstherefrom),

a CDR2 comprising the sequence RASELQW (SEQ ID NO: 7) (or a sequencethat differs by no more than, 1, 2, or 3 amino acids therefrom), and

a CDR3 comprising the sequence QRSNEVPWT (SEQ ID NO: 8) (or a sequencethat differs by no more than, 1, 2, or 3 amino acids therefrom).

In some embodiments, V of HCDR1 is unchanged; in some embodiments, L ofHCDR2 is unchanged, and in some embodiments, both V of HCDR 1 and L ofHCDR2 are unchanged.

In certain embodiments, the antibody molecule comprises a VH FW1 havingthe sequence QVQLVQSGAEVKKPGASVKVSCKAGFNIK (SEQ ID NO: 11), or an aminoacid sequence having no more than 1, 2, 3, 4, or 5 mutations relative toSEQ ID NO: 11.

In certain embodiments, the antibody molecule comprises a VH FW2 havingthe sequence WVRQAPGQGLEWMG (SEQ ID NO: 84), or an amino acid sequencehaving no more than 1, 2, 3, 4, or 5 mutations relative to SEQ ID NO:84. In certain embodiments, the antibody molecule comprises a VH FW2having the sequence WVRQAPEQGLEWMG (SEQ ID NO: 85), or an amino acidsequence having no more than 1, 2, 3, 4, or 5 mutations relative to SEQID NO: 85.

In certain aspects, the present disclosure provides an antibody moleculecapable of binding dengue virus, comprising:

(a) a heavy chain immunoglobulin variable region segment comprising:

a FW1 comprising a deletion of position 26 relative to SEQ ID NO: 33;

a CDR1 comprising the sequence DTYMS (SEQ ID NO: 14) (or a sequence thatdiffers by no more than, 1, 2, or 3 amino acids therefrom, optionallyprovided that T is unchanged), or a CDR1 comprising the sequence DVYMS(SEQ ID NO: 3) (or a sequence that differs by no more than, 1, 2, or 3amino acids therefrom, optionally provided that V is unchanged),

a CDR2 comprising the sequence RIDPENGDTKYDPKLQG (SEQ ID NO: 4) (or asequence that differs by no more than, 1, 2, 3, 4, or 5 amino acidstherefrom, optionally provided that L is unchanged), and

a CDR3 comprising the sequence GWEGFAY (SEQ ID NO: 5) (or a sequencethat differs by no more than, 1, 2, or 3 amino acids therefrom); and

(b) a light chain variable region segment comprising:

a CDR1 comprising the sequence RASENVDKYGNSFMH (SEQ ID NO: 6) (or asequence that differs by no more than, 1, 2, 3, 4, or 5 amino acidstherefrom),

a CDR2 comprising the sequence RASELQW (SEQ ID NO: 7) (or a sequencethat differs by no more than, 1, 2, or 3 amino acids therefrom), and

a CDR3 comprising the sequence QRSNEVPWT (SEQ ID NO: 8) (or a sequencethat differs by no more than, 1, 2, or 3 amino acids therefrom).

In certain embodiments, the antibody molecule comprises a VH FW2 havingthe sequence WVRQAPGQGLEWMG (SEQ ID NO: 84), or an amino acid sequencehaving no more than 1, 2, 3, 4, or 5 mutations relative to SEQ ID NO:84. In certain embodiments, the antibody molecule comprises a VH FW2having the sequence WVRQAPEQGLEWMG (SEQ ID NO: 85), or an amino acidsequence having no more than 1, 2, 3, 4, or 5 mutations relative to SEQID NO: 85.

In certain aspects, the present disclosure provides an antibodymolecule, optionally capable of binding dengue virus, comprising:

(a) a heavy chain immunoglobulin variable region segment comprising:

a CDR1 comprising the sequence DVYMS (SEQ ID NO: 3) (or a sequence thatdiffers by no more than, 1, 2, or 3 amino acids therefrom, optionallyprovided that V is unchanged),

a CDR2 comprising the sequence RIDPENGDTKYDPKLQG (SEQ ID NO: 4) (or asequence that differs by no more than, 1, 2, 3, 4, or 5 amino acidstherefrom, optionally provided that L is unchanged), and

a CDR3 comprising the sequence GWEGFAY (SEQ ID NO: 5) (or a sequencethat differs by no more than, 1, 2, or 3 amino acids therefrom),optionally provided that A is replaced with I, K, D or E.

(b) a light chain variable region segment comprising:

a CDR1 comprising the sequence RASENVDKYGNSFMH (SEQ ID NO: 6) (or asequence that differs by no more than, 1, 2, 3, 4, or 5 amino acidstherefrom), optionally provided a Y is replaced with F,

a CDR2 comprising the sequence RASELQW (SEQ ID NO: 7) (or a sequencethat differs by no more than, 1, 2, or 3 amino acids therefrom), and

a CDR3 comprising the sequence QRSNEVPWT (SEQ ID NO: 8) (or a sequencethat differs by no more than, 1, 2, or 3 amino acids therefrom).

Accordingly, in some embodiments, the heavy chain CDR3 is GWEGFIY (SEQID NO: 90), GWEGFKY (SEQ ID NO: 91), GWEGFDY (SEQ ID NO: 92), or GWEGFEY(SEQ ID NO: 93). In some embodiments, the light chain CDR1 isRASENVDKFGNSFMH (SEQ ID NO: 94). Put another way, in some embodiments,position 105 of the heavy chain, which is alanine in antibody A11, maybe changed to another residue, e.g., an I, K, D or E. In certainembodiments, position 32 of the light chain, which is tyrosine inantibody A11, is changed to another residue, e.g., F. In someembodiments, a mutation described in this paragraph improves theantibody molecule's affinity for EDIII and/or its neutralizationactivity towards one or more (or all) strain or serotype of denguevirus.

In certain embodiments, the antibody molecule comprises a VH FW1 havingthe sequence QVQLVQSGAEVKKPGASVKVSCKAGFNIK (SEQ ID NO: 11), or an aminoacid sequence having no more than 1, 2, 3, 4, or 5 mutations relative toSEQ ID NO: 11.

In certain embodiments, the antibody molecule comprises a VH FW2 havingthe sequence WVRQAPGQGLEWMG (SEQ ID NO: 84), or an amino acid sequencehaving no more than 1, 2, 3, 4, or 5 mutations relative to SEQ ID NO:84. In certain embodiments, the antibody molecule comprises a VH FW2having the sequence WVRQAPEQGLEWMG (SEQ ID NO: 85), or an amino acidsequence having no more than 1, 2, 3, 4, or 5 mutations relative to SEQID NO: 85.

In some embodiments of the aspects herein, the antibody molecule iscapable of binding to dengue virus EDIII (E protein domain III). Incertain embodiments, the antibody molecule comprises one or more CDRshaving the sequence of any of SEQ ID NOS: 3-8, 14, and 35 (or a sequencethat differs by no more than, 1, 2, or 3 amino acids therefrom). Forexample, the antibody molecule may comprise at least two, three, four,five, or six CDRs having the sequence of any of SEQ ID NOS: 3-8, 14, and35.

In some embodiments, the antibody molecule comprises a VH CDR1 of SEQ IDNO: 3 or 14, a VH CDR2 of SEQ ID NO: 4 or 35, a VH CDR3 of SEQ ID NO: 5,a VL CDR1 of SEQ ID NO: 6, a VL CDR2 of SEQ ID NO: 7, and a VL CDR3 ofSEQ ID NO: 8. For instance, the antibody molecule may comprise a VH CDR1of SEQ ID NO: 3, a VH CDR2 of SEQ ID NO: 4, a VH CDR3 of SEQ ID NO: 5, aVL CDR1 of SEQ ID NO: 6, a VL CDR2 of SEQ ID NO: 7, and a VL CDR3 of SEQID NO: 8.

In some embodiments, the antibody molecule comprises a VH amino acidsequence at least 70%, 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQID NO: 1.

In some embodiments, the antibody molecule comprises a VH amino acidsequence at least 70%, 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQID NO: 80.

In some embodiments, the antibody molecule comprises a VH amino acidsequence at least 70%, 80%, 85%, 90%, 95%, 99%, or 100% identical to anyof SEQ ID NOs. 16-21, 24, 25, 27, 29, 31, 32, 33, 36, 80, or 81. In someembodiments, the antibody molecule comprises a VL amino acid sequence atleast 70%, 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 2 or34.

In certain embodiments, the antibody molecule is a Fab, F(ab′)2, Fv, ora single chain Fv fragment (scFv). In some embodiments, the antibodymolecule comprises a heavy chain constant region selected from IgG1,IgG2, IgG3, and IgG4. In some embodiments, the antibody moleculecomprises a light chain constant region chosen from the light chainconstant regions of kappa or lambda.

The antibody molecule may be an isolated antibody molecule and/or ahumanized antibody molecule. In some embodiments, the antibody moleculecontains one or more framework regions derived from a human frameworkgermline sequence.

In some embodiments, the antibody molecule is capable of binding todengue virus EDIII with a dissociation constant (K_(D)) of less thanabout 80, 70, 60, 50, 40, 30, 20, 10, 8, 6, 4, 3, 2, 1, 0.5, 0.2, or 0.1nM. The antibody molecule may be capable of binding to dengue virusserotype DV-4 EDIII with a dissociation constant (K_(D)) of less thanabout 10, 8, 6, 5, 4, or 3 nM. The antibody molecule may be capable ofbinding to DV-3 or DV-4 EDIII domain with at least a 2, 3, 4, 5, 6, 8,10, 12, 100, 1,000-fold greater affinity than antibody A11 or antibody4E11. The antibody molecule may be capable of binding to a dengue virusstrain chosen from one or more of DENV-4 BC2, DENV-4-Sing10, DENV-4NewCal09, DENV-4 Phil56, DENV-3 Sing09, DENV-3 Nic10, DENV-3 H87, DENV-2Peru95, DENV-2 Sing08, DENV-2 NGC, DENV-1 Hawaii/1944, DENV-2 NewGuinea/1944 (NGC), DENV-3 Philippines/1956 (H87), DENV-4 Mexico/1997(BC287/97), and DENV-4 H241, with at least 2, 3, 4, 5, 6, 8, 10, 12, 25,50, 75, 100, or 1,000-fold greater affinity than antibody A11 orantibody 4E11. The antibody molecule may be capable of neutralizingdengue virus in a focus reduction neutralization test or a related testfor evaluating neutralization of viral activity. The antibody moleculemay be capable of neutralizing dengue virus with an IC50 that is atleast 2, 3, 4, 5, 6, 8, 10, 12, 50, 75, or 100-fold lower than antibodyA11 or antibody 4E11 in a focus reduction neutralization test or arelated test for evaluating neutralization of viral activity.

In some aspects, the present disclosure provides a pharmaceuticalcomposition comprising the antibody molecule of any of the above claimsand a pharmaceutically acceptable carrier, excipient, or stabilizer.

The present disclosure also provides, e.g., a nucleic acid encoding theantibody heavy or light chain variable region of an antibody molecule asdescribed herein. The disclosure also provides, for example, expressionvector comprising such a nucleic acid. The disclosure also provides, forexample, a host cell comprising such a nucleic acid. The presentdisclosure additionally provides, e.g., a method of producing anantibody molecule or fragment thereof as described herein, comprisingculturing the host cell under conditions suitable for gene expression.

In some aspects, this disclosure provides a kit comprising an antibodymolecule as described herein. The kit may comprise a container, and thecontainer may have the antibody molecule disposed therein. The kit mayalso comprise a pharmaceutically acceptable carrier, excipient, orstabilizer, optionally admixed with the antibody molecule. The kit mayalso comprise a delivery device, e.g., one comprising a syringe orneedle. The kit may also comprise instructions for use.

In certain aspects, the present disclosure provides a method ofneutralizing dengue virus, comprising: contacting the dengue virus withan antibody molecule as described herein. In some embodiments, thedengue virus is of serotype DV-1, DV-2, DV-3, or DV-4.

In some aspects, the present disclosure provides a method of treating adengue virus infection, comprising administering to a subject in needthereof an isolated antibody molecule as described herein, in an amounteffective to treat the virus. The method may further compriseadministering an anti-viral agent to the subject, e.g., an anti-viralagent chosen from one or more of balapiravir, chloroquine, celgosivir,ivermectin, or Carica folia. In certain embodiments, the antiviral agentis a second anti-dengue antibody molecule, e.g., an anti-dengue antibodymolecule described herein different from a first anti-dengue antibodymolecule. In other embodiments, the antiviral agent is selected from analpha-glucosidase I inhibitor (e.g., celgosivir), an adenosinenucleoside inhibitor (e.g., NITD008); an RNA-dependent RNA polymerase(RdRp) inhibitor (e.g., NITD107), an inhibitor of host pyrimidinebiosynthesis, e.g., host dihydroorotate dehydrogenase (DHODH) (e.g.,NITD-982 and brequinar), an inhibitor of viral NS4B protein (e.g.,NITD-618), and an iminosugar (e.g., UV-4). The method may furthercomprise administering a vaccine to the subject, e.g., a dengue virusvaccine. In some embodiments, administration of the antibody molecule isparenteral or intravenous.

The disclosure also provides prophylactic methods. In some embodiments,a method of preventing a dengue virus infection by administering anantibody molecule as disclosed herein to a subject who is not, at thetime, infected with dengue virus. For instance, in certain aspects, thepresent disclosure provides a method of reducing a patient's risk ofcontracting dengue virus, comprising administering to a subject in needthereof an isolated antibody molecule as described herein, in an amounteffective to reduce the risk of contracting the virus. For example therisk of contracting dengue virus may be reduced by, e.g., at least 25%,50%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or more. In someembodiments, the antibody molecule is provided to a patient who is notinfected with dengue virus, with the result that if infection occurs,the course of the disease is likely to be milder than the course ofdisease in a similar patient who has not received the antibody molecule.For instance, the antibody molecule may reduce the risk of dengue feverdeveloping (e.g., the patient is more likely to experience anasymptomatic infection). The risk of dengue fever developing may bereduced, e.g., by at least 25%, 50%, 75%, 80%, 85%, 90%, 95%, 97%, 98%,99%, pr more compared to a patient that did not receive the antibodymolecule. In some embodiments, the risk of dengue fever progressing intodengue hemorrhagic fever may be reduced, e.g., by at least 25%, 50%,75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or more compared to a patientthat did not receive the antibody molecule.

The disclosure also provides methods of reducing or preventingtransmission of dengue virus (e.g., reducing or preventing transmissionbetween a subject (e.g., a human) and a mosquito. In certainembodiments, the subject is infected with dengue virus. In otherembodiments, the subject is not, at the time, infected with denguevirus, but is at risk of dengue viral infection. For instance, incertain aspects, the present disclosure provides a method of reducing orpreventing transmission of dengue virus (e.g., reducing or preventingtransmission of dengue virus between a subject (e.g., human) and amosquito), comprising administering to a subject an isolated antibodymolecule as described herein, in an amount effective to reduce thetransmission of dengue virus. For example, the transmission of denguevirus, e.g., from a subject to a mosquito, can be reduced by, e.g., atleast about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, compared tothe transmission from a subject that did not receive the antibodymolecule, e.g., as measured by a mosquito model described herein. As aresult, in some embodiments, the transmission of dengue virus from aninfected mosquito to an uninfected subject (e.g., human) can be furtherreduced, e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or90%.

In certain aspects, this disclosure provides a method of detectingdengue virus in a biological sample, comprising (i) contacting thesample or the subject (and optionally, a reference sample or subject)with an antibody molecule described herein under conditions that allowinteraction of the antibody molecule and the polypeptide to occur, and(ii) detecting formation of a complex between the antibody molecule andthe sample or the subject (and optionally, the reference sample orsubject).

In some aspects, this disclosure provides an anti-dengue antibodymolecule comprising a VH region that has a deletion of position 26relative to the VH of antibody A11. For instance, the anti-dengueantibody molecule of claim may have a VH region with between about 1-30,5-30, 10-30, 15-30, or 20-25 mutations relative to a VH of antibody A11.

In some aspects, this disclosure provides an antibody molecule capableof binding to dengue virus, which comprises a VH CDR1 having thesequence of SEQ ID NO: 3, or an amino acid sequence having no more than1, 2, 3, 4, 5, 10, or 15 mutations relative to SEQ ID NO: 3. In someembodiments, the mutations are substitutions, e.g., conservativesubstitutions.

In certain aspects, this disclosure provides an antibody moleculecapable of binding to dengue virus, which comprises a VH FW1 having thesequence QVQLVQSGAEVKKPGASVKVSCKAGFNIK (SEQ ID NO: 11), or an amino acidsequence having no more than 1, 2, 3, 4, 5, 10, or 15 mutations relativeto SEQ ID NO: 11. In some embodiments, the mutations are independentlyselected from deletions and substitutions, e.g., conservativesubstitutions. An antibody molecule of this paragraph may also have thefeatures described throughout this application, e.g., in the previousparagraph.

In some aspects, this disclosure provides antibody molecules capable ofbinding to dengue virus, which comprises a VH CDR2 having the sequenceof SEQ ID NO: 4, or an amino acid sequence having no more than 1, 2, 3,4, 5, 10, or 15 mutations relative to SEQ ID NO: 4. In some embodiments,the mutations are substitutions, e.g., conservative substitutions. Anantibody molecule of this paragraph may also have the features describedthroughout this application, e.g., in the previous two paragraphs.

In certain aspects, this disclosure provides an antibody moleculecapable of binding to dengue virus, which comprises a VH FW2 having thesequence WVRQAPGQGLEWMG (SEQ ID NO: 84) or WVRQAPEQGLEWMG (SEQ ID NO:85), or an amino acid sequence having no more than 1, 2, 3, 4, 5, or 10mutations relative to SEQ ID NO: 84 or SEQ ID NO: 85. In someembodiments, the mutations are independently selected from deletions andsubstitutions, e.g., conservative substitutions. An antibody molecule ofthis paragraph may also have the features described throughout thisapplication, e.g., in the previous paragraph.

In some embodiments, the antibody molecule is capable of binding toEDIII of two or more, e.g., three or four, dengue virus serotypes, e.g.,selected from DV-1, DV-2, DV-3, and DV-4, with a dissociation constant(K_(D)) of less than about 80, 70, 60, 50, 40, 30, 20, 10, 8, 6, 4, 3,2, 1, 0.5, 0.2, 0.1, 0.05, or 0.01 nM for each of said two or moreserotypes.

In some embodiments, the antibody molecule has a variable region that isidentical in sequence, or which differs by 1, 2, 3, or 4 amino acid froma variable region described herein (e.g., an FR region disclosedherein).

In some embodiments, the anti-dengue antibody molecule is a fullantibody or fragment thereof (e.g., a Fab, F(ab′)2, Fv, or a singlechain Fv fragment (scFv)). In certain embodiments, the anti-dengueantibody molecule is a monoclonal antibody or an antibody with singlespecificity. The anti-dengue antibody molecule can also be a humanized,chimeric, camelid, shark, or in vitro-generated antibody molecule. Insome embodiments, the anti-dengue antibody molecule thereof is ahumanized antibody molecule. The heavy and light chains of theanti-dengue antibody molecule can be full-length (e.g., an antibody caninclude at least one or at least two complete heavy chains, and at leastone or at least two complete light chains) or can include anantigen-binding fragment (e.g., a Fab, F(ab′)2, Fv, a single chain Fvfragment, a single domain antibody, a diabody (dAb), a bivalent orbispecific antibody or fragment thereof, a single domain variantthereof, or a camelid antibody).

In certain embodiments, the anti-dengue antibody molecule has a heavychain constant region (Fc) chosen from, e.g., the heavy chain constantregions of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE;particularly, chosen from, e.g., the heavy chain constant regions ofIgG1, IgG2, IgG3, and IgG4, more particularly, the heavy chain constantregion of IgG1 or IgG2 (e.g., human IgG1 or IgG2). In some embodiments,the heavy chain constant region is human IgG1. In some embodiments, theanti-dengue antibody molecule has a light chain constant region chosenfrom, e.g., the light chain constant regions of kappa or lambda, in someembodiments kappa (e.g., human kappa). In some embodiments, the constantregion is altered, e.g., mutated, to modify the properties of theanti-dengue antibody molecule (e.g., to increase or decrease one or moreof: Fc receptor binding, antibody glycosylation, the number of cysteineresidues, effector cell function, or complement function). For example,the constant region may be mutated at positions 234 (e.g., L to A), 235(e.g., L to A), 296 (e.g., M to Y), 297 (e.g., N to A or G or Q), 298(e.g., S to T), 300 (e.g., T to E), 477 (e.g., H to K) and 478 (e.g., Nto F) to alter Fc receptor binding.

In some embodiments, the antibody molecule is a humanized antibodymolecule.

In some embodiments, the antibody molecule is isolated or recombinant.

In some embodiments, the anti-dengue antibody molecules comprisecombinations of human or humanized framework regions with CDRs(complementarity determining regions).

The present disclosure also provides nucleic acids comprising nucleotidesequences that encode heavy and light chain variable regions and CDRs ofthe anti-dengue antibody molecules, as described herein. For example,the disclosure provides a first and second nucleic acid encoding heavyand light chain variable regions, respectively, of an anti-dengueantibody molecule according to Table 1, e.g., D88, A48, F38, F108, orC88, or a sequence substantially identical thereto. For example, thenucleic acid can comprise a nucleotide sequence encoding an anti-dengueantibody molecule according to Table 1, e.g., D88, A48, F38, F108, orC88, or a sequence substantially identical to that nucleotide sequence(e.g., a sequence at least about 85%, 90%, 95%, 99% or more identicalthereto, or which differs by no more than 3, 6, 15, 30, or 45nucleotides from the aforementioned nucleotide sequence). In certainembodiments, the nucleic acid can comprise a nucleotide sequenceencoding at least one, two, or three CDRs from a heavy chain variableregion having an amino acid sequence as set forth in Table 2, or asequence substantially homologous thereto (e.g., a sequence at leastabout.85%, 90%, 95%, 99% or more identical thereto, and/or having one,two, three or more substitutions, insertions or deletions, e.g.,conserved substitutions). In certain embodiments, the nucleic acid cancomprise a nucleotide sequence encoding at least one, two, or three CDRsfrom a light chain variable region having an amino acid sequence as setforth in Table 2, or a sequence substantially homologous thereto (e.g.,a sequence at least about 85%, 90%, 95%, 99% or more identical thereto,and/or having one, two, three or more substitutions, insertions ordeletions, e.g., conserved substitutions). In some embodiments, thenucleic acid can comprise a nucleotide sequence encoding at least one,two, three, four, five, or six CDRs from heavy and light chain variableregions having an amino acid sequence as set forth in Table 2, or asequence substantially homologous thereto (e.g., a sequence at leastabout 85%, 90%, 95%, 99% or more identical thereto, and/or having one,two, three or more mutations (e.g., substitutions, insertions ordeletions, e.g., conserved substitutions). In some embodiments, anucleic acid having a structural feature discussed in this paragraphencodes an antibody molecule or portion thereof having one or moreadvantageous properties such as an improved (e.g., relative to A11)affinity for or neutralization activity towards dengue virus, e.g.,DV-4. In some embodiments, the advantageous property is a property ofList 1, e.g., one or more (e.g., two, three, four, five, six, seven,eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen,or all) of properties (a), (b), (c), (d), (e), (m), (n), (o), (p), (q),(r), (s), (t), (u), (v), (w), or (x).

This disclosure also provides nucleic acid sequences, e.g., a nucleicacid that hybridizes to a nucleic acid sequence that encodes an antibodyof Table 1, e.g., D88, A48, F38, F108, or C88, and its complement, e.g.,under low stringency, medium stringency, or high stringency, or otherhybridization condition described herein. This application alsodiscloses nucleic acid sequences of Table 4, and their complements,e.g., under low stringency, medium stringency, or high stringency, orother hybridization condition described herein. In some embodiments, thenucleic acid is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% orhigher identical to a sequence of Table 4, its complement, or a portionof any of the aforementioned sequences. In some embodiments, a nucleicacid having a structural feature discussed in this paragraph encodes anantibody molecule or portion thereof having one or more advantageousproperties such as an improved (e.g., relative to A11) affinity for orneutralization activity towards dengue virus, e.g., DV-4. In someembodiments, the advantageous property is a property of List 1, e.g.,one or more (e.g., two, three, four, five, six, seven, eight, nine, ten,eleven, twelve, thirteen, fourteen, fifteen, sixteen, or all) ofproperties (a), (b), (c), (d), (e), (m), (n), (o), (p), (q), (r), (s),(t), (u), (v), (w), or (x).

In certain aspects, this disclosure features host cells and vectorscontaining the nucleic acids described herein. The nucleic acids may bepresent in a single vector or separate vectors present in the same hostcell or separate host cell. The host cell can be a eukaryotic cell,e.g., a mammalian cell, an insect cell, a yeast cell, or a prokaryoticcell, e.g., E. coli. For example, the mammalian cell can be a culturedcell or a cell line. Exemplary mammalian cells include human cells e.g.,HEK293 cells, lymphocytic cell lines (e.g., NS0), Chinese hamster ovarycells (CHO), COS cells, oocyte cells, and cells from a transgenicanimal.

In some aspects, the present disclosure provides a method of providingan antibody molecule described herein. The method may include: providingan antibody molecule that specifically binds to an EDIII antigen; makingone or more mutations to the antibody (e.g., to the constant region,framework, and/or CDRs) and evaluating if the antibody moleculespecifically binds to the EDIII antigen, or evaluating efficacy of theantibody molecule in inhibiting dengue virus function. The method canfurther include purifying the antibody molecule. For example, theantibody molecule can be purified by one or more chromatography stepscomprising, e.g., affinity chromatography, e.g., Protein Achromatography. The method can further include administering theantibody molecule to a subject, e.g., a human or non-human animal.

In certain aspects, the disclosure provides, compositions, e.g.,pharmaceutical compositions, which include a pharmaceutically acceptablecarrier, excipient or stabilizer, and at least one of the anti-dengueantibody molecules described herein. In some embodiments, the antibodymolecule is conjugated to a label or a therapeutic agent. In someembodiments, the compositions, e.g., the pharmaceutical compositions,comprise a combination of the antibody molecule and a second agent,e.g., a therapeutic agent, or two or more of the aforesaid antibodymolecules, as further described herein.

The antibody molecules disclosed herein can inhibit one or moreactivities of dengue virus. For instance, the antibody molecules maydisrupt the native structure of the E protein on the surface of thevirion, e.g., causing inactivation of the virus. As a result, theantibody molecule may neutralize the virus, inhibit its ability to entera host cell, or reduce viral stability. In some embodiments, an antibodymolecule neutralizes dengue virus (e.g., in a focus reductionneutralization test or a related test for evaluating neutralization ofviral activity) with an EC50 or FRNT50 of less than or equal to 1400,1000, 800, 600, 550, 500, 450, 400, 350, 300, 350, 200, 150, 100, 50, or25 ng/ml. In some embodiments, the antibody neutralizes dengue virus(e.g., in a focus reduction neutralization test or a related test forevaluating neutralization of viral activity) with an IC50 of less thanor equal to 20, 17.6, 15, 10, 5, 4, 2, 1.4, 1, or 0.50 μg/mL. In someembodiments, neutralization of DV-4 is tested, and in some embodiments,neutralization of one of DV-1, DV-2, or DV-3 is tested.

The subject can be a mammal, e.g., a monkey, a primate, preferably ahigher primate, e.g., a human (e.g., a patient having, or at risk ofhaving, dengue virus).

This disclosure also provides a method of treating dengue virus in asubject, comprising administering to the subject an anti-dengue antibodymolecule_(s) described herein, e.g., a therapeutically effective amountof an anti-dengue antibody molecule, or an antigen-binding portionthereof. In some embodiments, the anti-dengue antibody molecule isadministered to a patient suffering from dengue virus, resulting in thereduction of viral load and/or the reduction of at least one symptom,e.g., selected from sudden-onset fever, headache, muscle and jointpains, weight loss, central nervous system penetration, and/or rash. Insome embodiments, the anti-dengue antibody molecule is administered to apatient at risk of being infected from dengue virus, resulting in thereduction of the risk of becoming infected or reduction of the severityof the infection if infection does occur.

The anti-dengue antibody molecule can be administered to the subjectsystemically (e.g., orally, parenterally, subcutaneously, intravenously,rectally, intramuscularly, intraperitoneally, intranasally,transdermally, or by inhalation or intracavitary installation),topically, or by application to mucous membranes, such as the nose,throat and bronchial tubes.

The methods and compositions described herein can be used in combinationwith other therapeutic modalities. In some embodiments, the methods ofdescribed herein include administering to the subject an anti-dengueantibody molecule as described herein, in combination with a secondtreatment or prophylactic for dengue virus, in an amount effective totreat or prevent said disorder. The antibody molecule and the secondagent can be administered simultaneously or sequentially.

Any combination and sequence of the anti-dengue antibody molecules andother therapeutic modalities can be used. The anti-dengue antibodymolecule and/or other therapeutic modalities can be administered duringperiods of active infection, or during a period of remission or lessactive disease. The anti-dengue antibody molecule and other therapeuticmodalities can be administered before treatment, concurrently withtreatment, post-treatment, or during remission of the infection.

In some aspects, the present disclosure provides methods for detectingthe presence of dengue virus in a sample, e.g., in vitro or in vivo(e.g., a biological sample, e.g., blood or serum). The methods hereincan be used to evaluate (e.g., monitor treatment or progression of,diagnose and/or stage a disorder described herein, e.g., dengue virus,in a subject). The method may include: (i) contacting the sample with(and optionally, a reference, e.g., a control sample), or administeringto the subject, an anti-dengue antibody molecule as described herein,under conditions that allow interaction to occur, and (ii) detectingwhether there is formation of a complex between the antibody moleculeand the sample (and optionally, the reference, e.g., control, sample).Formation of the complex is indicative of the presence of dengue virus,and can indicate the suitability or need for a treatment describedherein. The method can involve, e.g., an immunohistochemistry,immunocytochemistry, FACS, antibody molecule-complexed magnetic beads,ELISA assays, or PCR-techniques (e.g., RT-PCR).

Typically, the anti-dengue antibody molecule used in the in vivo and invitro diagnostic methods is directly or indirectly labeled with adetectable substance to facilitate detection of the bound or unboundbinding agent. Suitable detectable substances include variousbiologically active enzymes, prosthetic groups, fluorescent materials,luminescent materials, paramagnetic (e.g., nuclear magnetic resonanceactive) materials, and radioactive materials.

In some aspects, the present disclosure provides diagnostic ortherapeutic kits that include the anti-dengue antibody moleculesdescribed herein and instructions for use.

The disclosure contemplates all combinations of any one or more of theforegoing aspects and/or embodiments, as well as combinations with anyone or more of the embodiments set forth in the detailed description andexamples.

Other features, objects, and advantages of the compositions and methodsherein will be apparent from the description and drawings, and from theclaims.

Figures and Tables are provided herewith.

BRIEF DESCRIPTION OF THE DRAWINGS

Each of the Figures is described herein in more detail.

FIGS. 1A-1I show the amino acid and nucleotide sequences of severalanti-dengue EDIII antibodies. Kabat CDRs are underlined, and certainresidues of interest are boxed (shown with a gray background in thepriority documents).

FIG. 2 depicts the results of a focus reduction neutralization testusing live virus showing that antibody B11 improves upon mAb A11neutralization of DV4.

FIG. 3 shows a repeated assay of antibodies A11 and B11 neutralizingDV4.

FIG. 4 is a functional assessment of frameworks of several humanizedEDIII antibodies.

FIG. 5 shows antibody affinity upon back-mutations of the N-terminus ofanti-dengue antibodies. Heavy chain FW “04” refers to a type offramework that has the same heavy chain framework amino acid sequenceas, e.g., mAb B48. Light chain FW “08” refers to a type of frameworkthat has the same light chain framework amino acid sequence as, e.g.,mAb B48. The heavy and light chain framework sequences of mAb B48 areshown in Tables 1 and 2.

FIG. 6 shows that combining certain point mutations leads to improvedaffinity for EDIII. Light chain FW “08” refers to a type of frameworkthat has the same light chain framework amino acid sequence as, e.g.,mAb D88. The light chain framework sequence of mAb D88 is shown inTables 1 and 2.

FIG. 7 shows the results of setting position 98 to A, V, or S incombination with other mutations.

FIG. 8 shows the results of competition ELISA to determine the EDIIIbinding affinity of select antibodies.

FIG. 9 shows the binding of selected anti-dengue antibodies to EDIII offour dengue virus serotypes.

FIG. 10A summarizes the phylogenetic relationship of the EDIII aminoacid sequences of selected dengue virus isolates. “DENV” is anabbreviation for dengue virus, and DENV-2 represents serotype DV-2,DENV-3 represents serotype DV-3, and DENV-4 represents serotype DV-4.

FIG. 10B shows the binding of antibody D88 to a panel of diverse denguevirus isolates. * indicates strain used for in vitro neutralizationtesting.

FIG. 11 shows the affinity of selected anti-dengue antibodies forvarious strains of dengue virus.

FIG. 12 shows the ability of several anti-dengue antibodies toneutralize dengue virus serotype DV-4 in a focus reductionneutralization test.

FIG. 13 shows the ability of several anti-dengue antibodies toneutralize dengue virus serotype DV-3 in a focus reductionneutralization test. DV-3 H87 was used in this test.

FIG. 14 shows the ability of several anti-dengue antibodies toneutralize dengue virus serotype DV-4 in a focus reductionneutralization test.

FIGS. 15A-15C show the thermal stability of selected anti-dengueantibodies based on a thermal shift analysis assay (Sypro Orange).

FIG. 16 shows the effect of antibody D88 on survival of mice infectedwith dengue virus in an AG129 mouse model.

FIG. 17 shows the effect of antibody D88 on weight change of miceinfected with dengue virus in an AG129 mouse model.

FIG. 18 shows the effect of antibody D88 on viremia in mice infectedwith dengue virus serotype 2 (strain NGC) in an AG129 mouse model.

FIG. 19 shows the ability of antibody D88 to neutralize dengue virusserotypes DENV-1, DENV-2, DENV-3, and DENV-4 propagated in C6/36 insectcells in a focus reduction neutralization test (FRNT).

FIG. 20 shows the ability of antibody D88 to neutralize dengue virusserotypes DENV-1, DENV-2, DENV-3, and DENV-4 propagated in Vero (monkey)cells in a focus reduction neutralization test (FRNT).

FIG. 21 shows the ability of antibody D88 and A11 to neutralize denguevirus DENV-4 strain H241 propagated in Vero (monkey) cells in a focusreduction neutralization test (FRNT).

FIG. 22 shows the aggregation propensity of antibodies A48, C88 and D88as evaluated by high performance size exclusion chromatography (HP-SEC).

FIG. 23 depicts the affinity gain of antibody D88 to DENV-4 withconcurrent improved affinity to DENV-1, DENV-2 and DENV-3 compared toantibody 4E11.

BRIEF DESCRIPTION OF THE TABLES

Each of the Tables is described herein in more detail.

Table 1 summarizes the sequences of exemplary anti-dengue antibodies.

Table 2 depicts the amino acid sequences of the heavy chain variabledomain and light chain variable domain sequences of Table 1. Kabat CDRsare underlined, Chothia CDRs are italicized, and certain residues ofinterest are shown with a gray background.

Table 3 depicts the amino acid sequences of the CDRs of Table 1.

Table 4 summarizes the nucleic acid sequences encoding the antibodies ofTable 1.

Table 5 depicts the nucleic acid sequences summarized in Table 4.

Table 6 depicts additional amino acid sequences described throughout theapplication.

DETAILED DESCRIPTION

Disclosed herein are antibody molecules that bind to dengue virusepitopes, e.g., EDIII, with high affinity and specificity.Advantageously, several of the antibody molecules herein bind with highaffinity to EDIII of dengue virus serotypes DV-1, DV-2, DV-3, and DV-4.Nucleic acid molecules encoding the antibody molecules, expressionvectors, host cells and methods for making the antibody molecules arealso provided. The anti-dengue antibody molecules disclosed herein canbe used (alone or in combination with other agents or therapeuticmodalities) to treat, prevent and/or diagnose dengue virus, e.g., DV-1,DV-2, DV-3, or DV-4. As used herein, DV-1, DV-2, DV-3, and DV-4 aresometimes referred to as DENV-1, DENV-2, DENV-3, and DENV-4,respectively.

Definitions

As used herein, the articles “a” and “an” refer to one or to more thanone (e.g., to at least one) of the grammatical object of the article.

The term “or” is used herein to mean, and is used interchangeably with,the term “and/or”, unless context clearly indicates otherwise.

“About” and “approximately” shall generally mean an acceptable degree oferror for the quantity measured given the nature or precision of themeasurements. Exemplary degrees of error are within 20 percent (%),typically, within 10%, and more typically, within 5% of a given value orrange of values.

The compositions and methods disclosed herein encompass polypeptides andnucleic acids having the sequences specified, or sequences substantiallyidentical or similar thereto, e.g., sequences at least 85%, 90%, 95%identical or higher to the sequence specified. In the context of anamino acid sequence, the term “substantially identical” is used hereinto refer to a first amino acid that contains a sufficient or minimumnumber of amino acid residues that are i) identical to, or ii)conservative substitutions of aligned amino acid residues in a secondamino acid sequence such that the first and second amino acid sequencescan have a common structural domain and/or common functional activity.For example, amino acid sequences that contain a common structuraldomain having at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% identity to a reference sequence, e.g., a sequenceprovided herein.

In the context of nucleotide sequence, the term “substantiallyidentical” is used herein to refer to a first nucleic acid sequence thatcontains a sufficient or minimum number of nucleotides that areidentical to aligned nucleotides in a second nucleic acid sequence suchthat the first and second nucleotide sequences encode a polypeptidehaving common functional activity, or encode a common structuralpolypeptide domain or a common functional polypeptide activity. Forexample, nucleotide sequences having at least about 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% identity to a reference sequence,e.g., a sequence provided herein.

The term “functional variant” refers polypeptides that have asubstantially identical amino acid sequence to the naturally-occurringsequence, or are encoded by a substantially identical nucleotidesequence, and are capable of having one or more activities of thenaturally-occurring sequence.

To determine the percent identity of two amino acid sequences, or of twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in one or both of a first and asecond amino acid or nucleic acid sequence for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes). Ina preferred embodiment, the length of a reference sequence aligned forcomparison purposes is at least 30%, e.g., at least 40%, 50%, 60%, e.g.,at least 70%, 80%, 90%, 100% of the length of the reference sequence.The amino acid residues or nucleotides at corresponding amino acidpositions or nucleotide positions are then compared. When a position inthe first sequence is occupied by the same amino acid residue ornucleotide as the corresponding position in the second sequence, thenthe molecules are identical at that position.

The percent identity between the two sequences is a function of thenumber of identical positions shared by the sequences, taking intoaccount the number of gaps, and the length of each gap, which need to beintroduced for optimal alignment of the two sequences.

The comparison of sequences and-determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. In some embodiments, the percent identity between two aminoacid sequences is determined using the Needleman and Wunsch ((1970) J.Mol. Biol. 48:444-453) algorithm which has been incorporated into theGAP program in the GCG software package (available athttp://www.gcg.com), using either a Blossum 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6. In certain embodiments, the percentidentity between two nucleotide sequences is determined using the GAPprogram in the GCG software package (available at http://www.gcg.com),using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80and a length weight of 1, 2, 3, 4, 5, or 6. One suitable set ofparameters (and the one that should be used unless otherwise specified)are a Blossum 62 scoring matrix with a gap penalty of 12, a gap extendpenalty of 4, and a frameshift gap penalty of 5.

The percent identity between two amino acid or nucleotide sequences canbe determined using the algorithm of E. Meyers and W. Miller ((1989)CABIOS, 4:11-17) which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4.

The nucleic acid and protein sequences described herein can be used as a“query sequence” to perform a search against public databases to, forexample, identify other family members or related sequences. Suchsearches can be performed using the NBLAST and XBLAST programs (version2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLASTnucleotide searches can be performed with the NBLAST program, score=100,wordlength=12 to obtain nucleotide sequences homologous to a nucleicacid as described herein. BLAST protein searches can be performed withthe XBLAST program, score=50, wordlength=3 to obtain amino acidsequences homologous to protein molecules described herein. To obtaingapped alignments for comparison purposes, Gapped BLAST can be utilizedas described in Altschul et al., (1997) Nucleic Acids Res. 25:3389-3402.When utilizing BLAST and gapped BLAST programs, the default parametersof the respective programs (e.g., XBLAST and NBLAST) can be used. Seewww.ncbi.nlm.nih.gov.

As used herein, the term “hybridizes under low stringency, mediumstringency, high stringency, or very high stringency conditions”describes conditions for hybridization and washing. Guidance forperforming hybridization reactions can be found in Current Protocols inMolecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6, which isincorporated by reference. Aqueous and nonaqueous methods are describedin that reference and either can be used. Specific hybridizationconditions referred to herein are as follows: 1) low stringencyhybridization conditions in 6× sodium chloride/sodium citrate (SSC) atabout 45° C., followed by two washes in 0.2×SSC, 0.1% SDS at least at50° C. (the temperature of the washes can be increased to 55° C. for lowstringency conditions); 2) medium stringency hybridization conditions in6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1%SDS at 60° C.; 3) high stringency hybridization conditions in 6×SSC atabout 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 65°C.; and preferably 4) very high stringency hybridization conditions are0.5M sodium phosphate, 7% SDS at 65° C., followed by one or more washesat 0.2×SSC, 1% SDS at 65° C. Very high stringency conditions (4) aresuitable conditions and the ones that should be used unless otherwisespecified.

It is understood that the molecules described herein may have additionalconservative or non-essential amino acid substitutions, which do nothave a substantial effect on their functions.

A “conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., lysine, arginine, histidine), acidic side chains(e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine).

The terms “polypeptide,” “peptide” and “protein” (if single chain) areused interchangeably herein.

The terms “nucleic acid,” “nucleic acid sequence,” “nucleotidesequence,” or “polynucleotide sequence,” and “polynucleotide” are usedinterchangeably.

The term “isolated,” as used herein, refers to material that is removedfrom its original or native environment (e.g., the natural environmentif it is naturally occurring). For example, a naturally-occurringpolynucleotide or polypeptide present in a living animal is notisolated, but the same polynucleotide or polypeptide, separated by humanintervention from some or all of the co-existing materials in thenatural system, is isolated. Such polynucleotides could be part of avector and/or such polynucleotides or polypeptides could be part of acomposition, and still be isolated in that such vector or composition isnot part of the environment in which it is found in nature.

As used herein, the term “treat”, e.g., a dengue virus infection, meansthat a subject (e.g., a human) who has been infected with a virus andexperiences symptoms of the virus, will, in embodiments, suffer lesssevere symptoms and/or will recover faster when the antibody molecule isadministered than if the antibody were never administered. Inembodiments, when an infection is treated, an assay to detect virus inthe subject will detect less virus after effective treatment for theinfection. For example, a diagnostic assay using an antibody molecule,such as an antibody molecule described herein, will detect less or novirus in a biological sample of a patient after administration of anantibody molecule for the effective treatment of the infection. Otherassays, such as PCR (e.g., qPCR) can also be used to monitor treatmentin a patient, to detect the presence, e.g., decreased presence (orabsence) after treatment of viral infection in the patient. Treatmentcan, e.g., partially or completely alleviate, ameliorate, relieve,inhibit, reduce the severity of, and/or reduce incidence and optionally,delay onset of, one or more manifestations of the effects or symptoms,features, and/or causes of a particular disease, disorder, and/orcondition (e.g., dengue virus). In embodiments treatment is of a subjectwho does not exhibit certain signs of the relevant disease, disorderand/or condition and/or of a subject who exhibits only early signs ofthe disease, disorder, and/or condition. In embodiments treatment is ofa subject who exhibits one or more established signs of the relevantdisease, disorder and/or condition. In embodiments, treatment is of asubject diagnosed as suffering from dengue virus.

As used herein, the term “prevent”, e.g., a dengue virus infection,means that a subject (e.g., a human) is less likely to be infected by avirus (e.g., dengue virus) if the subject receives the antibody prior to(e.g., 1 day, 2 days, 1 week, 2 weeks, 3 weeks, or 1 month of more)being exposed to the virus.

As used herein, the terms “framework,” “FW” and “FR” are usedinterchangeably and have identical meaning in this document and itspriority documents.

Various aspects of the compositions and methods herein are described infurther detail below. Additional definitions are set out throughout thespecification.

Anti-Dengue Antibody Molecules

Exemplary sequences of anti-dengue antibodies are described in Tables1-4 below.

TABLE 1 Summary of the amino acid sequences of exemplary anti-dengueantibodies. Antibody designation SEQ ID NO Description D88 1 VH aminoacid sequence 2 VL amino acid sequence 3 VH CDR1 amino acid sequence,Kabat 4 VH CDR2 amino acid sequence, Kabat 5 VH CDR3 amino acidsequence, Kabat 6 VL CDR1 amino acid sequence, Kabat 7 VL CDR2 aminoacid sequence, Kabat 8 VL CDR3 amino acid sequence, Kabat 9 VH CDR1amino acid sequence, Chothia 10 VH CDR2 amino acid sequence, Chothia 5VH CDR3 amino acid sequence, Chothia 6 VL CDR1 amino acid sequence,Chothia 7 VL CDR2 amino acid sequence, Chothia 8 VL CDR3 amino acidsequence, Chothia 11 VH FW1 amino acid sequence, Kabat 85 VH FW2 aminoacid sequence, Kabat F38 80 VH amino acid sequence 2 VL amino acidsequence 3 VH CDR1 amino acid sequence, Kabat 4 VH CDR2 amino acidsequence, Kabat 5 VH CDR3 amino acid sequence, Kabat 6 VL CDR1 aminoacid sequence, Kabat 7 VL CDR2 amino acid sequence, Kabat 8 VL CDR3amino acid sequence, Kabat 9 VH CDR1 amino acid sequence, Chothia 10 VHCDR2 amino acid sequence, Chothia 5 VH CDR3 amino acid sequence, Chothia6 VL CDR1 amino acid sequence, Chothia 7 VL CDR2 amino acid sequence,Chothia 8 VL CDR3 amino acid sequence, Chothia 11 VH FW1 amino acidsequence, Kabat 84 VH FW2 amino acid sequence, Kabat A48 16 VH aminoacid sequence 2 VL amino acid sequence 14 VH CDR1 amino acid sequence,Kabat 4 VH CDR2 amino acid sequence, Kabat 5 VH CDR3 amino acidsequence, Kabat 6 VL CDR1 amino acid sequence, Kabat 7 VL CDR2 aminoacid sequence, Kabat 8 VL CDR3 amino acid sequence, Kabat 15 VH CDR1amino acid sequence, Chothia 10 VH CDR2 amino acid sequence, Chothia 5VH CDR3 amino acid sequence, Chothia 6 VL CDR1 amino acid sequence,Chothia 7 VL CDR2 amino acid sequence, Chothia 8 VL CDR3 amino acidsequence, Chothia C88 17 VH amino acid sequence 2 VL amino acid sequence3 VH CDR1 amino acid sequerice, Kabat 4 VH CDR2 amino acid sequence,Kabat 5 VH CDR3 amino acid sequence, Kabat 6 VL CDR1 amino acidsequence, Kabat 7 VL CDR2 amino acid sequence, Kabat 8 VL CDR3 aminoacid sequence, Kabat 9 VH CDR1 amino acid sequence, Chothia 10 VH CDR2amino acid sequence, Chothia 5 VH CDR3 amino acid sequence, Chothia 6 VLCDR1 amino acid sequence, Chothia 7 VL CDR2 amino acid sequence, Chothia8 VL CDR3 amino acid sequence, Chothia 85 VH FW2 amino acid sequence,Kabat F108 81 VH amino acid sequence 2 VL amino acid sequence 3 VH CDR1amino acid sequence, Kabat 4 VH CDR2 amino acid sequence, Kabat 5 VHCDR3 amino acid sequence, Kabat 6 VL CDR1 amino acid sequence, Kabat 7VL CDR2 amino acid sequence, Kabat 8 VL CDR3 amino acid sequence, Kabat9 VH CDR1 amino acid sequence, Chothia 10 VH CDR2 amino acid sequence,Chothia 5 VH CDR3 amino acid sequence, Chothia 6 VL CDR1 amino acidsequence, Chothia 7 VL CDR2 amino acid sequence, Chothia 8 VL CDR3 aminoacid sequence, Chothia 84 VH FW2 amino acid sequence, Kabat B48 18 VHamino acid sequence 2 VL amino acid sequence 14 VH CDR1 amino acidsequence, Kabat 4 VH CDR2 amino acid sequence, Kabat 5 VH CDR3 aminoacid sequence, Kabat 6 VL CDR1 amino acid sequence, Kabat 7 VL CDR2amino acid sequence, Kabat 8 VL CDR3 amino acid sequence, Kabat 15 VHCDR1 amino acid sequence, Chothia 10 VH CDR2 amino acid sequence,Chothia 5 VH CDR3 amino acid sequence, Chothia 6 VL CDR1 amino acidsequence, Chothia 7 VL CDR2 amino acid sequence, Chothia 8 VL CDR3 aminoacid sequence, Chothia A68 19 VH amino acid sequence 2 VL amino acidsequence 14 VH CDR1 amino acid sequence, Kabat 4 VH CDR2 amino acidsequence, Kabat 5 VH CDR3 amino acid sequence, Kabat 6 VL CDR1 aminoacid sequence, Kabat 7 VL CDR2 amino acid sequence, Kabat 8 VL CDR3amino acid sequence, Kabat 15 VH CDR1 amino acid sequence, Chothia 10 VHCDR2 amino acid sequence, Chothia 5 VH CDR3 amino acid sequence, Chothia6 VL CDR1 amino acid sequence, Chothia 7 VL CDR2 amino acid sequence,Chothia 8 VL CDR3 amino acid sequence, Chothia A100 20 VH amino acidsequence 2 VL amino acid sequence 14 VH CDR1 amino acid sequence, Kabat4 VH CDR2 amino acid sequence, Kabat 5 VH CDR3 amino acid sequence,Kabat 6 VL CDR1 amino acid sequence, Kabat 7 VL CDR2 amino acidsequence, Kabat 8 VL CDR3 amino acid sequence, Kabat 15 VH CDR1 aminoacid sequence, Chothia 10 VH CDR2 amino acid sequence, Chothia 5 VH CDR3amino acid sequence, Chothia 6 VL CDR1 amino acid sequence, Chothia 7 VLCDR2 amino acid sequence, Chothia 8 VL CDR3 amino acid sequence, ChothiaC58 21 VH amino acid sequence 2 VL amino acid sequence 14 VH CDR1 aminoacid sequence, Kabat 4 VH CDR2 amino acid sequence, Kabat 5 VH CDR3amino acid sequence, Kabat 6 VL CDR1 amino acid sequence, Kabat 7 VLCDR2 amino acid sequence, Kabat 8 VL CDR3 amino acid sequence, Kabat 22VH CDR1 amino acid sequence, Chothia 10 VH CDR2 amino acid sequence,Chothia 5 VH CDR3 amino acid sequence, Chothia 6 VL CDR1 amino acidsequence, Chothia 7 VL CDR2 amino acid sequence, Chothia 8 VL CDR3 aminoacid sequence, Chothia C78 23 VH amino acid sequence 2 VL amino acidsequence 3 VH CDR1 amino acid sequence, Kabat 4 VII CDR2 amino acidsequence, Kabat 5 VH CDR3 amino acid sequence, Kabat 6 VL CDR1 aminoacid sequence, Kabat 7 VL CDR2 amino acid sequence, Kabat 8 VL CDR3amino acid sequence, Kabat 24 VH CDR1 amino acid sequence, Chothia 10 VHCDR2 amino acid sequence, Chothia 5 VH CDR3 amino acid sequence, Chothia6 VL CDR1 amino acid sequence, Chothia 7 VL CDR2 amino acid sequence,Chothia 8 VL CDR3 amino acid sequence, Chothia C68 25 VH amino acidsequence 2 VL amino acid sequence 3 VH CDR1 amino acid sequence, Kabat 4VH CDR2 amino acid sequence, Kabat 5 VH CDR3 amino acid sequence, Kabat6 VL CDR1 amino acid sequence, Kabat 7 VL CDR2 amino acid sequence,Kabat 8 VL CDR3 amino acid sequence, Kabat 26 VH CDR1 amino acidsequence, Chothia 10 VH CDR2 amino acid sequence, Chothia 5 VH CDR3amino acid sequence, Chothia 6 VL CDR1 amino acid sequence, Chothia 7 VLCDR2 amino acid sequence, Chothia 8 VL CDR3 amino acid sequence, ChothiaD98 27 VH amino acid sequence 2 VL amino acid sequence 14 VH CDR1 aminoacid sequence, Kabat 4 VH CDR2 amino acid sequence, Kabat 5 VH CDR3amino acid sequence, Kabat 6 VL CDR1 amino acid sequence, Kabat 7 VLCDR2 amino acid sequence, Kabat 8 VL CDR3 amino acid sequence, Kabat 28VH CDR1 amino acid sequence, Chothia 10 VH CDR2 amino acid sequence,Chothia 5 VH CDR3 amino acid sequence, Chothia 6 VL CDR1 amino acidsequence, Chothia 7 VL CDR2 amino acid sequence, Chothia 8 VL CDR3 aminoacid sequence, Chothia D188 29 VH amino acid sequence 2 VL amino acidsequence 3 VH CDR1 amino acid sequence, Kabat 4 VH CDR2 amino acidsequence, Kabat 5 VH CDR3 amino acid sequence, Kabat 6 VL CDR1 aminoacid sequence, Kabat 7 VL CDR2 amino acid sequence, Kabat 8 VL CDR3amino acid sequence, Kabat 30 VH CDR1 amino acid sequence, Chothia 10 VHCDR2 amino acid sequence, Chothia 5 VH CDR3 amino acid sequence, Chothia6 VL CDR1 amino acid sequence, Chothia 7 VL CDR2 amino acid sequence,Chothia 8 VL CDR3 amino acid sequence, Chothia C128 31 VH amino acidsequence 2 VL amino acid sequence 3 VH CDR1 amino acid sequence, Kabat 4VH CDR2 amino acid sequence, Kabat 5 VH CDR3 amino acid sequence, Kabat6 VL CDR1 amino acid sequence, Kabat 7 VL CDR2 amino acid sequence,Kabat 8 VL CDR3 amino acid sequence, Kabat 9 VH CDR1 amino acidsequence, Chothia 10 VH CDR2 amino acid sequence, Chothia 5 VH CDR3amino acid sequence, Chothia 6 VL CDR1 amino acid sequence, Chothia 7 VLCDR2 amino acid sequence, Chothia 8 VL CDR3 amino acid sequence, ChothiaC98 32 VH amino acid sequence 2 VL amino acid sequence 3 VH CDR1 aminoacid sequence, Kabat 4 VH CDR2 amino acid sequence, Kabat 5 VH CDR3amino acid sequence, Kabat 6 VL CDR1 amino acid sequence, Kabat 7 VLCDR2 amino acid sequence, Kabat 8 VL CDR3 amino acid sequence, Kabat 9VH CDR1 amino acid sequence, Chothia 10 VH CDR2 amino acid sequence,Chothia 5 VH CDR3 amino acid sequence, Chothia 6 VL CDR1 amino acidsequence, Chothia 7 VL CDR2 amino acid sequence, Chothia 8 VL CDR3 aminoacid sequence, Chothia A11 33 VH amino acid sequence 34 VL amino acidsequence 14 VH CDR1 amino acid sequence, Kabat 35 VH CDR2 amino acidsequence, Kabat 5 VH CDR3 amino acid sequence, Kabat 6 VL CDR1 aminoacid sequence, Kabat 7 VL CDR2 amino acid sequence, Kabat 8 VL CDR3amino acid sequence, Kabat 15 VH CDR1 amino acid sequence, Chothia 10 VHCDR2 amino acid sequence, Chothia 5 VH CDR3 amino acid sequence, Chothia6 VL CDR1 amino acid sequence, Chothia 7 VL CDR2 amino acid sequence,Chothia 8 VL CDR3 amino acid sequence, Chothia B11 36 VH amino acidsequence 34 VL amino acid sequence 14 VH CDR1 amino acid sequence, Kabat21 VH CDR2 amino acid sequence, Kabat 5 VH CDR3 amino acid sequence,Kabat 6 VL CDR1 amino acid sequence, Kabat 7 VL CDR2 amino acidsequence, Kabat 8 VL CDR3 amino acid sequence, Kabat 15 VH CDR1 aminoacid sequence, Chothia 10 VH CDR2 amino acid sequence, Chothia 5 VH CDR3amino acid sequence, Chothia 6 VL CDR1 amino acid sequence, Chothia 7 VLCDR2 amino acid sequence, Chothia 8 VL CDR3 amino acid sequence, Chothia

A11 and B11 are mouse antibodies, and the other, antibodies of Table 1are humanized antibodies.

TABLE 2 Depiction of the amino acid sequences of the heavy chain variable domain and light chainvariable domain sequences of Table 1. CDRs,defined according to the Kabat system, areunderlined and bold, while CDRs defined accordingto the Chothia system are italicized. Certainresidues of interest are shown with a graybackground. Deletions are indicated with a caret symbol ({circumflexover ( )}). SEQ ID Description NO Sequence D88 VH  1QVQLVQSGAEVKKPGASVKVSCKA{circumflex over ( )}GFNIK

WVRQAPEQGLEWMG

RVTMTADTSTNTAYMELRSLRSDDTAVYYCAR

WGQGTLVTVSS D88 VL  2 DIVMTQSPASLAVSLGERATISC

WYQQKPGQPPKLLI Y

GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC

FGQ GTKLEIK F38 VH 80 QVQLVQSGAEVKKPGASVKVSCKA{circumflex over ( )}GFNIK

WVRQAPGQGLEWMG

RVTMTADTSTNTAYMELRSLRSDDTAVYYCAR

WGQGTLVTVSS A48 VH 16 QVQLVQSGAEVKKPGASVKVSCKASGFNIK

WVRQAPEQGLEWMG

RVTMTADTSTNTAYMELRSLRSDDTAVYYCAR

WGQGTLVTVSS C88 VH 17 QVQLVQSGAEVKKPGASVKVSCKASGFNIK

WVRQAPEQGLEWMG

RVTMTADTSTNTAYMELRSLRSDDTAVYYCAR

WGQGTLVTVSS F108 VH 81 QVQLVQSGAEVKKPGASVKVSCKASGFNIK

WVRQAPGQGLEWMG

RVTMTADTSTNTAYMELRSLRSDDTAVYYCAR

WGQGTLVTVSS B48 VH 18 QVQLVQSGAEVKKPGASVKVSCKA{circumflex over ( )}GFNIK

WVRQAPEQGLEWMG

RVTMTADTSTNTAYMELRSLRSDDTAVYYCAR

WGQGTLVTVSS A68 VH 19 QVQLVQSGAEVKKPGASVKVSCKASGFNIK

WVRQAPEQGLEWMG

RVTMTADTSTNTAYMELRSLRSDDTAVYYCVR

WGQGTLVTVSS A100 VH 20 QVQLVQSGAEVKKPGASVKVSCKASGFNIK

WVRQAPEQGLEWMG

RVTMTADTSTNTAYMELRSLRSDDTAVYYCSR

WGQGTLVTVSS C58 VH 21 QVQLVQSGAEVKKPGASVKVSCKAS

NIK

WVRQAPEQGLEWMG

RVTMTADTSTNTAYMELRSLRSDDTAVYYCAR

WGQGTLVTVSS C78 VH 23 QVQLVQSGAEVKKPGASVKVSCKASGFNI

WVRQAPEQGLEWMG

RVTMTADTSTNTAYMELRSLRSDDTAVYYCAR

WGQGTLVTVSS C68 VH 25 QVQLVQSGAEVKKPGASVKVSCKASGFNI

WVRQAPEQGLEWMG

RVTMTADTSTNTAYMELRSLRSDDTAVYYCAR

WGQGTLVTVSS D98 VH 27 QVQLVQSGAEVKKPGASVKVSCKAS

FNIK

WVRQAPEQGLEWMG

RVTMTADTSTNTAYMELRSLRSDDTAVYYCAR

WGQGTLVTVSS D188 VH 29 QVQLVQSGAEVKKPGASVKVSCKASAFNIK

WVRQAPEQGLEWMG

RVTMTADTSTNTAYMELRSLRSDDTAVYYCAR

WGQGTLVTVSS C128 VH 31 QVQLVQSGAEVKKPGASVKVSCKASGFNIK

WVRQAPEQGLEWMG

RVTMTADTSTNTAYMELRSLRSDDTAVYYCSR

WGQGTLVTVSS C98 VH 32 QVQLVQSGAEVKKPGASVKVSCKASGFNIK

WVRQAPEQGLEWMG

RVTMTADTSTNTAYMELRSLRSDDTAVYYCVR

WGQGTLVTVSS A11 VH 33 QVKLLEQSGAELVKPGASVRLSCTASGFNIK

WVKQRPEQGLEWIG

KATITADTSSNTAYLHLSSLTSGDTAVYYCSR

WGQGTLVTVSA A11 VL 34 ELVMTQTPASLAVSLGQRATISC

WYQQKAGQPPKLLI Y

GIPARFSGSGSRTDFTLTINPVEADDVATYFC

FGG GTKLEIK B11 VH 36 QVKLLEQSGAELVKPGASVRLSCTA{circumflex over( )}GFNIK

WVKQRPEQGLEWIG

KATITADTSSNTAYLHLSSLTSGDTAVYYCSR

WGQGTLVTVSA

Throughout this application, reference is made to amino acid positionsbased on the variable region of mouse antibody A11. The variable regionof mouse antibody B11 has a deletion at position 26 relative to A11. Thehuman variable region sequences in Table 1 have a deletion of theglutamic acid sequence at position 6 of A11. Consequently, sequencesthat carry a deletion relative to A11 use a numbering system that isoffset. For example, A48 heavy chain has a deletion of the glutamic acidsequence at position 6 relative to A11. As a result, position 26 (aserine) of A48 VH is actually the twenty-fifth amino acid of the A48 VHsequence (SEQ ID NO: 16). As another example, D88 heavy chain has adeletion of the glutamic acid sequence at position 6 of A11 and adeletion of the serine at position 26 relative to A11. As a consequence,position 33 (a valine) of D88 VH, is actually the thirty-first aminoacid of the D88 VH sequence (SEQ ID NO: 1).

Some structural features of the antibodies can be noted based on the VHand VL sequences in Table 2. B11 has a deletion at position 26 relativeto the A11 VH region. D88 is a humanized antibody that has a deletion atposition 26 and a T33V mutation relative to the A48 VH region (remainingconsistent with A11 numbering). F38 is a humanized antibody that has adeletion at position 26 and T33V and E43G mutations relative to the A48VH region. A48 is a humanized antibody with the same CDRs as A11 . C88is a humanized antibody that has a T33V mutation relative to the A48 VHregion. F108 is a humanized antibody that has T33V and E43G mutationsrelative to the A48 VH region. B48 is a humanized antibody that has adeletion at position 26 relative to the A48 VH region. A68 is ahumanized antibody that has an A98V mutation relative to the A48 VHregion. A100 is a humanized antibody that has an A98S mutation relativeto the A48 VH region. C58 is a humanized antibody that has G27Y and F28Wmutations relative to the A48 VH region. C78 is a humanized antibodythat has K31Q and T33V mutations relative to the A48 VH region. C68 is ahumanized antibody that has K31S and T33V mutations relative to the A48VH region. D98 is a humanized antibody that has a G27A mutation relativeto the A48 VH region.

Other variations of the antibodies of Tables 1 and 2 are envisioned. Forinstance, this application provides antibody B48+A98V, which has an A98Vmutation relative to B48; A48+V2L, which has a V2L mutation relative toA48; A48+InsE6, which has an InsE6 mutation relative to A48; B48+V2L,which has a V2L mutation relative to B48; B48+InsE6, which has an InsE6mutation relative to B48; D118, which has F28W and T33V mutationsrelative to A48; D128, which has G27A, F28W, and T33V mutations relativeto A48; D138, which has G27Y, F28A, and T33V mutations relative to A48;D148, which has G27Y and T33V mutations relative to A48; D158, which hasG27Y, F28G, and T33V mutations relative to A48; D168, which has F28Y andT33V mutations relative to A48; C98, which has T33V and A98V mutationsrelative to A48; C128, which has T33V and A98S mutations relative toA48; D178, which has Del26, T33V, and A98V mutations relative to A48;and D188, which has Del26, T33V, and A98S mutations relative to A48.

TABLE 3 Depiction of the amino acid sequences of the CDRs of Table 1.SEQ ID NO Sequence  3 DVYMS  4 RIDPENGDTKYDPKLQG  5 GWEGFAY  6RASENVDKYGNSFMH  7 RASELQW  8 QRSNEVPWT  9 GFNIKDV 10 DPENGD 14 DTYMS 15GFNIKDT 22 YWNIKDT 24 GFNIQDV 26 GFNISDV 28 AFNIKDT 30 AFNIKDV 35RIDPENGDTKYDPKFQG

TABLE 4 Summary of the nucleic acid sequences encoding the antibodies ofTable 1. Antibody SEQ designation ID NO Description D88 37 VH nucleicacid sequence 38 VL nucleic acid sequence F38 82 VH nucleic acidsequence 38 VL nucleic acid sequence A48 39 VH nucleic acid sequence 38VL nucleic acid sequence C88 40 VH nucleic acid sequence 38 VL nucleicacid sequence F108 83 VH nucleic acid sequence 38 VL nucleic acidsequence B48 41 VH nucleic acid sequence 38 VL nucleic acid sequence A6842 VH nucleic acid sequence 38 VL nucleic acid sequence A100 86 VHnucleic acid sequence 38 VL nucleic acid sequence C58 43 VH nucleic acidsequence 38 VL nucleic acid sequence C78 44 VH nucleic acid sequence 38VL nucleic acid sequence C68 45 VH nucleic acid sequence 38 VL nucleicacid sequence D98 46 VH nucleic acid sequence 38 VL nucleic acidsequence D188 87 VH nucleic acid sequence 38 VL nucleic acid sequenceC128 88 VH nucleic acid sequence 38 VL nucleic acid sequence C98 89 VHnucleic acid sequence 38 VL nucleic acid sequence A11 47 VH nucleic acidsequence 48 VL nucleic acid sequence B11 49 VH nucleic acid sequence 48VL nucleic acid sequence

TABLE 5 Nucleic acid sequences of Table 4 SEQ ID NO Sequence 37CAAGTGCAACTCGTTCAGTCCGGAGCAGAAGTCAAGAAACCTGGAGCTTCAGTCAAAGTCAGCTGCAAGGCCGGCTTCAATATCAAGGACGTCTACATGTCCTGGGTGCGGCAGGCTCCAGAGCAAGGACTGGAATGGATGGGGCGCATTGACCCGGAGAACGGTGATACGAAGTACGACCCGAAACTGCAGGGCCGCGTGACCATGACCGCAGATACTAGCACCAACACCGCGTACATGGAGCTGCGGTCCTTGAGGTCGGATGACACTGCTGTGTATTACTGTGCCAGAGGCTGGGAAGGGTTCGCGTACTGGGGACAGGGAACTCTCGTGACTGTGTCGTCT 38GATATTGTCATGACCCAAAGCCCAGCCTCCCTCGCCGTGTCTCTCGGAGAAAGAGCAACTATCTCGTGCCGGGCTTCGGAGAATGTGGACAAGTACGGCAACTCCTTCATGCACTGGTACCAGCAGAAACCGGGACAGCCGCCTAAACTGTTGATCTACCGGGCGTCAGAACTGCAATGGGGAGTGCCTGACAGGTTTTCGGGTTCGGGATCCGGCACGGATTTCACCCTCACTATCTCCAGCCTGCAAGCAGAGGACGTTGCGGTGTACTACTGTCAGCGCTCAAACGAGGTCCCATGGACTTTTGGACAA GGGACCAAGCTGGAAATCAAG 82CAAGTGCAACTCGTTCAGTCCGGAGCAGAAGTCAAGAAACCTGGAGCTTCAGTCAAAGTCAGCTGCAAGGCCGGCTTCAATATCAAGGACGTCTACATGTCCTGGGTGCGGCAGGCTCCAGGGCAAGGACTGGAATGGATGGGGCGCATTGACCCGGAGAACGGTGATACGAAGTACGACCCGAAACTGCAGGGCCGCGTGACCATGACCGCAGATACTAGCACCAACACCGCGTACATGGAGCTGCGGTCCTTGAGGTCGGATGACACTGCTGTGTATTACTGTGCCAGAGGCTGGGAAGGGTTCGCGTACTGGGGACAGGGAACTCTCGTGACTGTGTCGTCT 39CAAGTGCAACTCGTTCAGTCCGGAGCAGAAGTCAAGAAACCTGGAGCTTCAGTCAAAGTCAGCTGCAAGGCCTCGGGCTTCAATATCAAGGACACCTACATGTCCTGGGTGCGGCAGGCTCCAGAGCAAGGACTGGAATGGATGGGGCGCATTGACCCGGAGAACGGTGATACGAAGTACGACCCGAAACTGCAGGGCCGCGTGACCATGACCGCAGATACTAGCACCAACACCGCGTACATGGAGCTGCGGTCCTTGAGGTCGGATGACACTGCTGTGTATTACTGTGCCAGAGGCTGGGAAGGGTTCGCGTACTGGGGACAGGGAACTCTCGTGACTGTGTCGTCT 40CAAGTGCAACTCGTTCAGTCCGGAGCAGAAGTCAAGAAACCTGGAGCTTCAGTCAAAGTCAGCTGCAAGGCCTCGGGCTTCAATATCAAGGACGTCTACATGTCCTGGGTGCGGCAGGCTCCAGAGCAAGGACTGGAATGGATGGGGCGCATTGACCCGGAGAACGGTGATACGAAGTACGACCCGAAACTGCAGGGCCGCGTGACCATGACCGCAGATACTAGCACCAACACCGCGTACATGGAGCTGCGGTCCTTGAGGTCGGATGACACTGCTGTGTATTACTGTGCCAGAGGCTGGGAAGGGTTCGCGTACTGGGGACAGGGAACTCTCGTGACTGTGTCGTCT 83CAAGTGCAACTCGTTCAGTCCGGAGCAGAAGTCAAGAAACCTGGAGCTTCAGTCAAAGTCAGCTGCAAGGCCTCGGGCTTCAATATCAAGGACGTCTACATGTCCTGGGTGCGGCAGGCTCCAGGGCAAGGACTGGAATGGATGGGGCGCATTGACCCGGAGAACGGTGATACGAAGTACGACCCGAAACTGCAGGGCCGCGTGACCATGACCGCAGATACTAGCACCAACACCGCGTACATGGAGCTGCGGTCCTTGAGGTCGGATGACACTGCTGTGTATTACTGTGCCAGAGGCTGGGAAGGGTTCGCGTACTGGGGACAGGGAACTCTCGTGACTGTGTCGTCT 41CAAGTGCAACTCGTTCAGTCCGGAGCAGAAGTCAAGAAACCTGGAGCTTCAGTCAAAGTCAGCTGCAAGGCCGGCTTCAATATCAAGGACACCTACATGTCCTGGGTGCGGCAGGCTCCAGAGCAAGGACTGGAATGGATGGGGCGCATTGACCCGGAGAACGGTGATACGAAGTACGACCCGAAACTGCAGGGCCGCGTGACCATGACCGCAGATACTAGCACCAACACCGCGTACATGGAGCTGCGGTCCTTGAGGTCGGATGACACTGCTGTGTATTACTGTGCCAGAGGCTGGGAAGGGTTCGCGTACTGGGGACAGGGAACTCTCGTGACTGTGTCGTCT 42CAAGTGCAACTCGTTCAGTCCGGAGCAGAAGTCAAGAAACCTGGAGCTTCAGTCAAAGTCAGCTGCAAGGCCTCGGGCTTCAATATCAAGGACACCTACATGTCCTGGGTGCGGCAGGCTCCAGAGCAAGGACTGGAATGGATGGGGCGCATTGACCCGGAGAACGGTGATACGAAGTACGACCCGAAACTGCAGGGCCGCGTGACCATGACCGCAGATACTAGCACCAACACCGCGTACATGGAGCTGCGGTCCTTGAGGTCGGATGACACTGCTGTGTATTACTGTGTCAGAGGCTGGGAAGGGTTCGCGTACTGGGGACAGGGAACTCTCGTGACTGTGTCGTCT 86CAAGTGCAACTCGTTCAGTCCGGAGCAGAAGTCAAGAAACCTGGAGCTTCAGTCAAAGTCAGCTGCAAGGCCTCGGGCTTCAATATCAAGGACACCTACATGTCCTGGGTGCGGCAGGCTCCAGAGCAAGGACTGGAATGGATGGGGCGCATTGACCCGGAGAACGGTGATACGAAGTACGACCCGAAACTGCAGGGCCGCGTGACCATGACCGCAGATACTAGCACCAACACCGCGTACATGGAGCTGCGGTCCTTGAGGTCGGATGACACTGCTGTGTATTACTGTTCCAGAGGCTGGGAAGGGTTCGCGTACTGGGGACAGGGAACTCTCGTGACTGTGTCGTCT 43CAAGTGCAACTCGTTCAGTCCGGAGCAGAAGTCAAGAAACCTGGAGCTTCAGTCAAAGTCAGCTGCAAGGCCTCGTACTGGAATATCAAGGACACCTACATGTCCTGGGTGCGGCAGGCTCCAGAGCAAGGACTGGAATGGATGGGGCGCATTGACCCGGAGAACGGTGATACGAAGTACGACCCGAAACTGCAGGGCCGCGTGACCATGACCGCAGATACTAGCACCAACACCGCGTACATGGAGCTGCGGTCCTTGAGGTCGGATGACACTGCTGTGTATTACTGTGCCAGAGGCTGGGAAGGGTTCGCGTACTGGGGACAGGGAACTCTCGTGACTGTGTCGTCT 44CAAGTGCAACTCGTTCAGTCCGGAGCAGAAGTCAAGAAACCTGGAGCTTCAGTCAAAGTCAGCTGCAAGGCCTCGGGCTTCAATATCCAGGACGTCTACATGTCCTGGGTGCGGCAGGCTCCAGAGCAAGGACTGGAATGGATGGGGCGCATTGACCCGGAGAACGGTGATACGAAGTACGACCCGAAACTGCAGGGCCGCGTGACCATGACCGCAGATACTAGCACCAACACCGCGTACATGGAGCTGCGGTCCTTGAGGTCGGATGACACTGCTGTGTATTACTGTGCCAGAGGCTGGGAAGGGTTCGCGTACTGGGGACAGGGAACTCTCGTGACTGTGTCGTCT 45CAAGTGCAACTCGTTCAGTCCGGAGCAGAAGTCAAGAAACCTGGAGCTTCAGTCAAAGTCAGCTGCAAGGCCTCGGGCTTCAATATCTCGGACGTCTACATGTCCTGGGTGCGGCAGGCTCCAGAGCAAGGACTGGAATGGATGGGGCGCATTGACCCGGAGAACGGTGATACGAAGTACGACCCGAAACTGCAGGGCCGCGTGACCATGACCGCAGATACTAGCACCAACACCGCGTACATGGAGCTGCGGTCCTTGAGGTCGGATGACACTGCTGTGTATTACTGTGCCAGAGGCTGGGAAGGGTTCGCGTACTGGGGACAGGGAACTCTCGTGACTGTGTCGTCT 46CAAGTGCAACTCGTTCAGTCCGGAGCAGAAGTCAAGAAACCTGGAGCTTCAGTCAAAGTCAGCTGCAAGGCCTCGGCCTTCAATATCAAGGACACCTACATGTCCTGGGTGCGGCAGGCTCCAGAGCAAGGACTGGAATGGATGGGGCGCATTGACCCGGAGAACGGTGATACGAAGTACGACCCGAAACTGCAGGGCCGCGTGACCATGACCGCAGATACTAGCACCAACACCGCGTACATGGAGCTGCGGTCCTTGAGGTCGGATGACACTGCTGTGTATTACTGTGCCAGAGGCTGGGAAGGGTTCGCGTACTGGGGACAGGGAACTCTCGTGACTGTGTCGTCT 87CAAGTGCAACTCGTTCAGTCCGGAGCAGAAGTCAAGAAACCTGGAGCTTCAGTCAAAGTCAGCTGCAAGGCCTCGGCCTTCAATATCAAGGACGTCTACATGTCCTGGGTGCGGCAGGCTCCAGAGCAAGGACTGGAATGGATGGGGCGCATTGACCCGGAGAACGGTGATACGAAGTACGACCCGAAACTGCAGGGCCGCGTGACCATGACCGCAGATACTAGCACCAACACCGCGTACATGGAGCTGCGGTCCTTGAGGTCGGATGACACTGCTGTGTATTACTGTGCCAGAGGCTGGGAAGGGTTCGCGTACTGGGGACAGGGAACTCTCGTGACTGTGTCGTCT 88CAAGTGCAACTCGTTCAGTCCGGAGCAGAAGTCAAGAAACCTGGAGCTTCAGTCAAAGTCAGCTGCAAGGCCTCGGGCTTCAATATCAAGGACGTCTACATGTCCTGGGTGCGGCAGGCTCCAGAGCAAGGACTGGAATGGATGGGGCGCATTGACCCGGAGAACGGTGATACGAAGTACGACCCGAAACTGCAGGGCCGCGTGACCATGACCGCAGATACTAGCACCAACACCGCGTACATGGAGCTGCGGTCCTTGAGGTCGGATGACACTGCTGTGTATTACTGTAGCAGAGGCTGGGAAGGGTTCGCGTACTGGGGACAGGGAACTCTCGTGACTGTGTCGTCT 89CAAGTGCAACTCGTTCAGTCCGGAGCAGAAGTCAAGAAACCTGGAGCTTCAGTCAAAGTCAGCTGCAAGGCCTCGGGCTTCAATATCAAGGACGTCTACATGTCCTGGGTGCGGCAGGCTCCAGAGCAAGGACTGGAATGGATGGGGCGCATTGACCCGGAGAACGGTGATACGAAGTACGACCCGAAACTGCAGGGCCGCGTGACCATGACCGCAGATACTAGCACCAACACCGCGTACATGGAGCTGCGGTCCTTGAGGTCGGATGACACTGCTGTGTATTACTGTGCCAGAGGCTGGGAAGGGTTCGCGTACTGGGGACAGGGAACTCTCGTGACTGTGTCGTCT 47CAAGTCAAACTGCTGGAACAGTCCGGAGCAGAGCTGGTGAAGCCTGGAGCGTCGGTGCGGCTTTCGTGTACCGCCTCCGGCTTTAACATCAAGGACACCTACATGTCGTGGGTGAAGCAGAGGCCCGAGCAGGGGCTCGAATGGATTGGCCGCATCGACCCGGAAAATGGTGATACCAAATACGACCCAAAGTTCCAGGGAAAGGCCACTATCACTGCAGATACTTCAAGCAACACCGCCTACCTCCACCTGTCCTCGCTCACTTCCGGAGATACCGCGGTCTACTATTGCTCAAGAGGATGGGAAGGCTTCGCGTACTGGGGTCAAGGAACGTTGGTGACCGTCAGCGCC 48GAATTGGTCATGACTCAGACGCCAGCTTCGCTGGCCGTGTCACTGGGACAGAGGGCCACTATCAGCTGCAGAGCATCGGAGAATGTGGATAAGTACGGGAACAGCTTCATGCACTGGTATCAACAGAAAGCTGGTCAACCTCCGAAGCTGCTTATCTACCGGGCGTCGGAACTCCAATGGGGCATTCCAGCACGGTTCAGCGGGTCGGGCTCCAGAACTGACTTCACCCTCACCATCAATCCCGTGGAGGCCGATGACGTGGCGACCTACTTTTGTCAGCGCTCCAACGAGGTCCCGTGGACTTTCGGAGGA GGAACCAAGCTGGAAATCAAG 49CAAGTCAAACTGCTGGAACAGTCCGGAGCAGAGCTGGTGAAGCCTGGAGCGTCGGTGCGGCTTTCGTGTACCGCCGGCTTTAACATCAAGGACACCTACATGTCGTGGGTGAAGCAGAGGCCCGAGCAGGGGCTCGAATGGATTGGCCGCATCGACCCGGAAAATGGTGATACCAAATACGACCCAAAGTTCCAGGGAAAGGCCACTATCACTGCAGATACTTCAAGCAACACCGCCTACCTCCACCTGTCCTCGCTCACTTCCGGAGATACCGCGGTCTACTATTGCTCAAGAGGATGGGAAGGCTTCGCGTACTGGGGTCAAGGAACGTTGGTGACCGTCAGCGCC

TABLE 6 Additional amino acid sequences. SEQ ID Description NO SequenceFW1 region of 11 QVQLVQSGAEVKKPGASVKV SEQ ID NO: 1 SCKAAGFNIKFW2 region of 84 WVRQAPGQGLEWMG SEQ ID NO: 80 FW2 region of 85WVRQAPEQGLEWMG SEQ ID NO: 1 EDIII-DV1 50 MTLKGMSYVMCTGSFKLEKEVAETQHGTVLVQVKYEGTDA PCKIPFSTQDEKGATQNGRL ITANPIVTDKEKPVNIEAEPPFGESYIVVGAGEKALKLSW FKKGSSIGK EDIII-DV2 51 MQLKGMSYSMCTGKFKVVKEIAETQHGTIVIRVQYEGDGS PCKIPFEIMDLEKRHVLGRL ITVNPIVTEKDSPVNIEAEPPFGDSYIIIGVEPGQLKLNW FKKGSSLE EDIII-DV3 52 MKLKGMSYAMCLNTFVLKKEVSETQHGTILIKVEYKGEDA PCKIPFSTEDGQGKAHNGRL ITANPVVTKKEEPVNIEAEPPFGESNIVIGIGDKALKINW YRKGSSIGK EDIII-DV4 53 MRIKGMSYTMCSGKFSIDKEMAETQHGTTVVKVKYEGAGA PCKVPIEIRDVNKEKVVGRI ISSTPLAENTNSVTNIELEPPFGDSYIVIGVGNSALTLHW FRKGSSIGK EDIII sequences fortesting breadth of binding ED3-DV1/Viet08 54 MTLKGMSYVMCTGSFKLEKELAETQHGTVLVQIKYEGTDA PCKIPFSTQDEKGVTQNGRL ITANPIVTDKEKPVNIEAEPPFGESYIVIGAGEKALKLSW FKKGSSIGK ED3- 55 MTLKGISYVMCTGPFKLEKEDV1/Malaysia05 VAETQHGTVLVQVKYEGTDA PCKIPFSSQDEKGVTQNGRLVTANPIVTDKEKPVNIEAEP PFGESYIVVGAGEKALKLSW FKKGSSIGK ED3- 56MTLKGTSYVMCTGSFKLEKE DV1/Mexico07 VAETQHGTVLVQVKYEGTDAPCKIPFSTQDEKGVTQNGRL ITANPIVTDKEKPVNIETEP PFGESYIVVGAGEKALKLSW FKKGSSIGKED3-DV2/Sing08 57 MQLKGMSYSMCTGKFKVVKE IAETQHGTIVIRVQYEGDGSPCKIPFEIMDLEKRHVLGRL ITVNPIVTEKDSPVNIEAEP PFGDSYIIIGVEPGQLKLSW FKKGSSIGQED3- 58 MQLKGMSYSMCTGKFKIVKE DV2/Venezuela07 IAETQHGTIVIRIQYEGDGSPCKIPFEITDLEKRHVLGRL ITVNPIVIEKDSPVNIEAEP PFGDSYIIIGVEPGQLKLNW FKKGSSIGQED3-DV2/Peru95 59 MQLKGMSYSMCTGKFKIVKE IAETQHGTIVIRVQYEGDGSPCKIPFEIMDLEKRHVLGRL ITVNPIVTEKDSPVNIEAEP PFGDSYIIIGVEPGQLKLDW FKKGSSIGQED3-DV2/Viet07 60 MQLKGMSYSMCTGKFKVVKE IAETQHGTIVIRVQYEGDGSPCKIPFEIMDLEKRYVLGRL ITVNPIVTEKDSPINIEAEP PFGDSYIIIGVEPGQLKLNW FKKGSSIGQED3- 61 MELKGMSYAMCLNTFVLKKE DV3/Cambodia08 VSETQHGTILIKVEYKGEDAPCKIPFSTEDGQGKAHSGRL ITANPVVTKKEEPVNIEAEP PFGESNIVIGIGDKALKINW YKKGSSIGKED3-DV3/Sing09 62 MELKGMSYAMCQNAFVLKKE VSETQHGTILIKVEYKGEDAPCKIPFSTEDGQGKAHNGRL ITANPVVTKKEEPVNIEAEP PFGESNIVIGIGDKALKINW YKKGSSIGKED3- 63 MELKGMSYAMCTNTFVLKKE DV3/Nicaragua10 VSETQHGTILIKVEYKGEDVPCKIPFSTEDGQGKAHNGRL ITANPVVIKKEEPVNIEAEP PFGESNIVIGIGDNALKINW YKKGSSIGKED3- 64 MELKGMSYAMCSGTFVLKKE DV3/PuertoRico77 VSETQHGTILIKIEYKGEDAPCKIPFSTEDAQGKAHNGRL ITANPVVTKKEEPVNIEAEP PFGESNIVIGTGDKALRINW YKKGSSIGKED3- 65 MRIKGMSYTMCSGKFSIDIC DV4/Venezuela08 EMAETQHMTVVKVKYEGAGAPCKVPIEIRDVNKEKVVGRV ISATPLAENTNSVTNIELEP PFGDSYIVIGVGNSALTLHW FRKGSSIGKED3-DV4/Sing10 66 MRIKGMSYTMCSGKFSIDKE MAETQHGTIVVKVKYEGAGAPCKVPIEIRDVNKEKVVGRI ESSTPFAENTNSVTNIELEP PFGDSYIVIGVGDSALTLHW FRKGSSIGKED3- 67 MRIKGMSYTMCSGKFSIDKE DV4/NewCal09 MAETQHGTTVVKVKYEGAGAPCKIPIEIRDVNKEKVVGRI ISSTPFAENTNSVINIELEP PFGDSYIVIGVGDSALTLHW FRKGSSIGKED3-DV4/ 68 MRIKGMSYTMCSGKFSIDKE Brazil11  MAETQHGTTVVKIKYEGTGAPCKVPIEIRDVNKEKVVGRI ISSTPFAENTNSVTNIELEP PFGDSYIVIGVGDSALTLHW FRKGSSIGKED3-DV4/Thai97 69 MRIKGMSYTMCSGKFSIDRE MAETQHGTTVVKVKYEGTGAPCKVPIEIRDVNKEKVVGRI ISSTPFAESTNSVTNIELEP PFGDSYIVIGVGDSALTLHW FRKGSSIGKED3- 70 MRIKGMSYTMCSGKFSIDKE DV4/H241/Phil56 MAETQHGTTVVKVKYEGAGAPCKVPIEIRDVNKEKVVGRI ISSTPFAEYTNSVTNIELEP PFGDSYIVIGVGDSALTLHW FRKGSSIGKHuman germline sequences, heavy chain Human germline = 71QVQLVQSGAEVKKPGSSVKV VH1-69, JH4 SCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANY AQKFQGRVTITADESTSTAY MELSSLRSEDTAVYYCARYFDYWGQGTLVTVSS Human germline = 72 QVQLVQSGAEVKKPGASVKV VH1-18, JH6SCKASGYTFTSYGISWVRQA PGQGLEWMGWISAYNGNTNY AQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARYM DVWGKGTTVTVSS Human germline = 73QVQLVQSGAEVKKPGASVKV VH1-18, JH4 SCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNTNY AQKLQGRVTMTTDTSTSTAY MELRSLRSDDTAVYYCARYMDVWGQGTLVTVSS Human germline = 74 EVQLVQSGAEVKKPGESLRI VH5-a*04, JH4SCKGSGYSFTSYWISWVRQM PGKGLEWMGRIDPSDSYTNY SPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARYM DVWGQGTLVTVSS Human germline = 75QVQLVQSGAEVKKPGASVKV VH1-46, JH4 SCKASGYTFNSYYMHWVRQAPGQGLEWMGIINPSGGSTSY AQKFQGRVTMTRDTSTSTVY MELSSLRSEDTAVYYCARYFDYWGQGTLVTVSS Human germline sequences, light chain Human Germline 76EIVLTQSPATLSLSPGERAT VK3D-11, Jk2 LSCRASQGVSSYLAWYQQKPGQAPRLLIYDASNRATGIPA RFSGSGPGTDFTLTISSLEP EDFAVYYCQQRSNWHCTFGQ GTKLEIKHuman Germline 77 DIQMTQSPSSLSASVGDRVT VK1-39, Jk4 ITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPS RFSGSGSGTDFTLTISSLQP EDFATYYCQQSYSFGGGTKV EIKHuman Germline 78 DIVMTQSPDSLAVSLGERAT VK4-1, Jk2 INCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTR ESGVPDRFSGSGSGTDFTLT ISSLQAEDVAVYYCQQYYSF GQGTKLEIKHuman Germline 79 DIVLTQSPASLAVSPGQRAT VK7-3 ITCRASESVSFLGINLIHWY(pseudogene), QQKPGQPPKLLIYQASNKDT Jk1 GVPARFSGSGSGTDFTLTINPVEANDTANYYCLQSKNFPW TFGQGTKVEIK

In some embodiments, the antibody molecule comprises a VH T33V mutationrelative to A11. More specifically, in some embodiments, the anti-dengueantibody molecule comprises the CDR1 of the VH region of an antibody ofTable 1 (e.g., D88, F38, F108, or C88), using the Kabat or Chothiadefinitions of CDRs. In some embodiments, the anti-dengue antibodymolecule comprises the CDR1 and one or both of CDR2 and CDR3 of the VHregion of an antibody of Table 1 (e.g., D88, A48, F38, F108, or C88),using the Kabat or Chothia definitions of CDRs. In some embodiments, theanti-dengue antibody molecule comprises CDR 1 of the VH region of anantibody of Table 1 (e.g., D88, A48, F38, F108, or C88) in combinationwith another 1, 2, 3, 4, or 5 (e.g., collectively 6) CDRs found in a VHand/or VL region of Table 2, using the Kabat of Chothia definitions ofCDRs. In some embodiments, the anti-dengue antibody molecule comprisesthe VH CDR1 of SEQ ID NO: 3. For instance, the anti-dengue antibodymolecule may comprise the VH CDR1 of SEQ ID NO: 3 in combination with aVH CDR2 and/or VHCDR3 of Table 3, e.g., VH CDR2 of SEQ ID NO: 4 and VHCDR3 of SEQ ID NO: 5. As a further example, the anti-dengue antibodymolecule may comprises the VH CDR 1 of SEQ ID NO: 3 in combination withanother 1, 2, 3, 4, or 5 (e.g., collectively 6) CDRs found in a VHand/or VL region of Table 2.

In certain embodiments, the antibody molecule comprises a VH F65Lmutation relative to A11. In a Kabat-defined CDR of A11, position 65 isa CDR residue, while in a Chothia-defined CDR of A11, position 65 is aframework residue. In some embodiments, an antibody molecule's affinityfor dengue virus is unaffected by the F65L mutation. In someembodiments, the anti-dengue antibody molecule comprises the CDR2 of theVH region of an antibody of Table 1 (e.g., D88, A48, F38, F108, or C88),using the Kabat or Chothia definitions of CDRs. In some embodiments, theanti-dengue antibody molecule comprises the CDR2 and one or both of CDR1and CDR3 of the VH region of an antibody of Table 1 (e.g., D88, A48,F38, F108, or C88), using the Kabat or Chothia definitions of CDRs. Insome embodiments, the anti-dengue antibody molecule comprises CDR2 ofthe VH region of an antibody of Table 1 (e.g., D88, A48, F38, F108, orC88) in combination with another 1, 2, 3, 4, or 5 (e.g., collectively 6)CDRs found in a VH and/or VL region of Table 2, using the Kabat ofChothia definitions of CDRs. In some embodiments, the anti-dengueantibody molecule comprises the VH CDR2 of SEQ ID NO: 4. For instance,the anti-dengue antibody molecule may comprise the VH CDR2 of SEQ ID NO:4 in combination with a VH CDR1 and/or VH CDR3 of Table 3, e.g., VH CDR1of SEQ ID NO: 3 and VH CDR3 of SEQ ID NO: 5. As a further example, theanti-dengue antibody molecule may comprises the VH CDR2 of SEQ ID NO: 4in combination with another 1, 2, 3, 4, or 5 (e.g., collectively. 6)CDRs found in a VH and/or VL region of Table 2. In certain embodiments,the antibody molecule comprises a VH F65L mutation and a VH T33Vmutation relative to A11.

In some embodiments, the anti-dengue antibody molecule comprises adeletion of the S (del26) at position 26 in the VH relative to A11. Insome embodiments, the antibody molecule comprises del26 mutation incombination with a VH T33V mutation and/or a VH F65L mutation. Incertain embodiments, the antibody molecule comprises a del26 mutationand one or more CDRs of Table 3. In certain embodiments, the antibodymolecule comprises a del26 mutation in combination with 1, 2, 3, 4, 5,or 6 CDRs, in a VH and/or VL region of Table 2, using the Kabat ofChothia definitions of CDRs.

As shown in Example 4 below, the N-terminus of the heavy chain istolerant to mutations. Accordingly, in some embodiments, positions 1-6of the heavy chain sequence have 1, 2, 3, 4, 5, or 6 mutations relativeto an antibody of Table 1. In some embodiments, an antibody molecule hasa substitution, insertion, or deletion at one or more (e.g., all) ofresidues 2, 3, 5, or 6 of a heavy chain sequence in Table 2. In certainembodiments, the antibody molecule comprises a portion of a heavy chainsequence of Table 2, e.g., amino acid positions 2-117, 3-117, 4-117,5-117, 6-117, 8-117, or 10-117.

As shown in Example 5 below, positions 27 and 28 in the VH are tolerantof mutations, and in some embodiments, a mutation to position 27 and/or28 enhances binding. Accordingly, in some embodiments, one or both ofpositions 27 and 28 have a mutation relative to an antibody of Table 1.

Example 5 also shows that position 98 in the VH is tolerant ofmutations, and in some embodiments, a mutation to position 98 enhancesbinding. Accordingly, in some embodiments, position 98 has a mutationrelative to an antibody of Table 1.

In some embodiments, the anti-dengue antibody molecule comprises a heavychain constant region, a light chain constant region, and heavy andlight chain variable regions of Table 2. In certain embodiments, theanti-dengue antibody molecule comprises a heavy chain constant region, alight chain constant region, and variable regions that comprise 1, 2, 3,4, 5, or 6 CDRs of Table 3.

In some embodiments, the heavy chain variable region is a heavy chainvariable region of Table 1, wherein residue 98 in the VH can be anyamino acid. In certain embodiments, residue 98 can be any unchargedamino acid. In some embodiments, position 98 can be A, V, or S. Example5 below shows that antibodies having residue A, V, or S at position 98have good binding to EDIII.

During the humanization process, various framework regions (e.g., VHFW1) can be back-mutated to contain residues from mouse antibodies A11or B11. More broadly, in some embodiments, the anti-dengue antibodymolecule comprises the sequence of all or a portion of a VH region ofTable 1. For instance, in some embodiments, the anti-dengue antibodymolecule comprises amino acids 5-117, 10-117, 15-117, 20-117, 25-117,30-117, or 32-117 of a VH region of Table 1. In some embodiments, theanti-dengue antibody molecule comprises a VH FW1 region selected from amouse VH FW1 region (e.g., that found in A11 or B11) or a human VH FW1region (e.g., one found in an antibody of Table 1 or a human germline VHFW1 sequence). In some embodiments, the VH FW1 region has no more than1, 2, 3, or 4 positions of non-identity relative to amino acids 1-31 ofa VH sequence of Table 1.

In some embodiments, the anti-dengue antibody molecule comprises a VHFW2 region of an antibody of Table 1. In some embodiments, the VH FW2region has no more than 1, 2, 3, or 4 positions of non-identity relativeto amino acids 37-50 of a VH sequence of Table 1. An antibody moleculecapable of cross-reacting with EDIII from more than one serotype ofdengue virus has several advantageous properties. For example, onetherapy can be used to treat or diagnose multiple serotypes of dengue.In addition, a physician need not determine which serotype infected apatient in order to determine the appropriate therapy. Accordingly, insome embodiments, the anti-dengue antibody molecule is capable ofindependently binding to two, three, four, or more dengue virusserotypes with high affinity. For instance, the antibody molecule mayindependently bind with high affinity to EDIII of DV-1 and DV-2; of DV-1and DV-3; of DV-1 and DV-4; of DV-2 and DV-3; of DV-2 and DV-4; of DV-3and DV-4; or DV-1 and DV-2 and DV-3; of DV-1 and DV-2 and DV-4; of DV-1and DV-3 and DV-4; of DV-2 and DV-3 and DV-4; or of DV-1 and DV-2 andDV-3 and DV-4. In certain embodiments, the antibody molecule canindependently bind with high affinity to EDIII of DV-4 and EDIII of oneor more other DV serotypes.

Each serotype of dengue virus mentioned above is a class containingnumerous strains. The antibody molecules described herein show a goodbreadth of reactivity, binding to multiple strains within differentserotypes (see FIG. 11). Accordingly, in some embodiments, an antibodymolecule as described herein binds to and/or neutralizes one or more(e.g., at least 2, 3, 4, 5, 10, 15, or 20, 25, or 30 or more) denguevirus strains, e.g., strains selected from: DENV-4 BC2, DENV-4-Sing10,DENV-4 NewCal09, DENV-4 Phil56, DENV-3 Sing09, DENV-3 Nic10, DENV-3 H87,DENV-2 Peru95, DENV-2 Sing08, DENV-2 NGC, DENV-1 Hawaii/1944, DENV-2 NewGuinea/1944 (NGC), DENV-3 Philippines/1956 (H87), DENV-4 Mexico/1997(BC287/97), and DENV-4 H241, the strains listed in the phylogenetic treeof FIG. 10A, the strains shown in FIGS. 10B and 19-21, the strainslisted in Table 6 herein (e.g., those strains for which EDIII sequencesare provided in Table 6), the strains deposited in the ATCC, the strainslisted in the World Reference Center for Emerging Viruses andArboviruses (WRCEVA) (available atwww.niaid.nih.gov/labsandresources/resources/dmid/wrceva/Pages/default.aspx),and the strains listed in the CDC's Division or Vector Borne InfectiousDiseases (available at www2a.cdc.gov/nczved/dvbid/misc/reg.asp).

In some embodiments, the antibody molecule binds with high affinity toone or more of DV-1, DV-2, DV-3, and DV-4. An EDIII amino acid sequenceof the E protein of each of these serotypes is, in some embodiments, anE protein sequence provided in Table 6.

In some embodiments, an antibody molecule disclosed herein does notactivate antibody-dependent enhancement (ADE). ADE is described in moredetail in Balsitis et al., Lethal Antibody Enhancement of Dengue Diseasein Mice Is Prevented by Fc Modification, PLoS Pathog 6(2):e1000790.doi:10.1371/journal.ppat.1000790. Briefly, ADE describes asituation in which a person experiences two sequential dengue infectionswith dengue viruses of different serotypes, and the occurrence of thefirst infection makes the second infection more severe (e.g., morelikely to progress into dengue hemorrhagic fever). A mechanism for ADEmay be that an anti-dengue antibody binds simultaneously to the virusand to an antibody Fc receptor on a host cell, increasing infectivity.As is clear from the FRNT experiments disclosed herein, this applicationprovides numerous antibody molecules that reduce, rather than increase,infectivity. Accordingly, in certain embodiments, an antibody moleculeas described herein does not activate ADE in a patient. In someembodiments, the antibody inhibits ADE that is induced by otherantibodies (e.g., the patient's endogenous antibodies).

In certain embodiments, the antibody molecule binds to a linear orconformational epitope on EDIII.

As used herein, the term “antibody molecule” refers to a proteincomprising at least one immunoglobulin variable domain sequence. Theterm antibody molecule includes, for example, full-length, matureantibodies and antigen-binding fragments of an antibody. For example, anantibody molecule can include a heavy (H) chain variable domain sequence(abbreviated herein as VH), and a light (L) chain variable domainsequence (abbreviated herein as VL). In another example, an antibodymolecule includes two heavy (H) chain variable domain sequences and twolight (L) chain variable domain sequence, thereby forming two antigenbinding sites, such as Fab, Fab′, F(ab′)2, Fc, Fd, Fd′, Fv, single chainantibodies (scFv for example), single variable domain antibodies,diabodies (Dab) (bivalent and bispecific), and chimeric (e.g.,humanized) antibodies, which may be produced by the modification ofwhole antibodies or those synthesized de novo using recombinant DNAtechnologies. These functional antibody fragments retain the ability toselectively bind with their respective antigen or receptor. Antibodiesand antibody fragments can be from any class of antibodies including,but not limited to, IgG, IgA, IgM, IgD, and IgE, and from any subclass(e.g., IgG1, IgG2, IgG3, and IgG4) of antibodies. The antibodies can bemonoclonal or polyclonal. The antibody can also be a human, humanized,CDR-grafted, or in vitro generated antibody. The antibody can have aheavy chain constant region chosen from, e.g., IgG1, IgG2, IgG3, orIgG4. The antibody can also have a light chain chosen from, e.g., kappaor lambda.

Examples of antigen-binding fragments include: (i) a Fab fragment, amonovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) aF(ab′)2 fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; (iii) a Fd fragmentconsisting of the VH and CH1 domains; (iv) a Fv fragment consisting ofthe VL and VH domains of a single arm of an antibody, (v) a diabody(dAb) fragment, which consists of a VH domain; (vi) a camelid orcamelized variable domain; (vii) a single chain Fv (scFv), see e.g.,Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc.Natl. Acad. Sci. USA 85:5879-5883); (viii) a single domain antibody.These antibody fragments may be obtained using any suitable method,including several conventional techniques known to those with skill inthe art, and the fragments can be screened for utility in the samemanner as are intact antibodies.

The term “antibody” includes intact molecules as well as functionalfragments thereof. Constant regions of the antibodies can be altered,e.g., mutated, to modify the properties of the antibody (e.g., toincrease or decrease one or more of: Fc receptor binding, antibodyglycosylation, the number of cysteine residues, effector cell function,or complement function).

The antibodies disclosed herein can also be single domain antibodies.Single domain antibodies can include antibodies whose complementarydetermining regions are part of a single domain polypeptide. Examplesinclude, but are not limited to, heavy chain antibodies, antibodiesnaturally devoid of light chains, single domain antibodies derived fromconventional 4-chain antibodies, engineered antibodies and single domainscaffolds other than those derived from antibodies. Single domainantibodies may be any of the art, or any future single domainantibodies. Single domain antibodies may be derived from any speciesincluding, but not limited to mouse, human, camel, llama, fish, shark,goat, rabbit, and bovine. According to some aspects, a single domainantibody is a naturally occurring single domain antibody known as heavychain antibody devoid of light chains. Such single domain antibodies aredisclosed in WO 9404678, for example. For clarity reasons, this variabledomain derived from a heavy chain antibody naturally devoid of lightchain is known herein as a VHH or nanobody to distinguish it from theconventional VH of four chain immunoglobulins. Such a VHH molecule canbe derived from antibodies raised in Camelidae species, for example incamel, llama, dromedary, alpaca and guanaco. Other species besidesCamelidae may produce heavy chain antibodies naturally devoid of lightchain; such VHHs are also contemplated.

The VH and VL regions can be subdivided into regions ofhypervariability, termed “complementarity determining regions” (CDR),interspersed with regions that are more conserved, termed “frameworkregions” (FR). The extent of the framework region and CDRs has beenprecisely defined by a number of methods (see, Kabat, E. A., et al.(1991) Sequences of Proteins of Immunological Interest, Fifth Edition,U.S. Department of Health and Human Services, NIH Publication No.91-3242; Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917; and theAbM definition used by Oxford Molecular's AbM antibody modelingsoftware. See, generally, e.g., Protein Sequence and Structure Analysisof Antibody Variable Domains. In: Antibody Engineering Lab Manual (Ed.:Duebel, S. and Kontermann, R., Springer-Verlag, Heidelberg). In someembodiments, the following definitions are used: AbM definition of CDR1of the heavy chain variable domain and Kabat definitions for the otherCDRs. In certain embodiments, Kabat definitions are used for all CDRs.In addition, embodiments described with respect to Kabat or AbM CDRs mayalso be implemented using Chothia hypervariable loops. Each VH and VLtypically includes three CDRs and four FRs, arranged from amino-terminusto carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4.

As used herein, an “immunoglobulin variable domain sequence” refers toan amino acid sequence which can form the structure of an immunoglobulinvariable domain. For example, the sequence may include all or part ofthe amino acid sequence of a naturally-occurring variable domain. Forexample, the sequence may or may not include one, two, or more N- orC-terminal amino acids, or may include other alterations that arecompatible with formation of the protein structure.

The term “antigen-binding region” refers to the part of an antibodymolecule that comprises determinants that form an interface that bindsto an E protein, or an epitope thereof. With respect to proteins (orprotein mimetics), the antigen-binding region typically includes one ormore loops (of at least, e.g., four amino acids or amino acid mimics)that form an interface that binds to the E protein. Typically, theantigen-binding region of an antibody molecule includes at least one ortwo CDRs, or more typically at least three, four, five or six CDRs.

The terms “monoclonal antibody” or “monoclonal antibody composition” asused herein refer to a preparation of antibody molecules of singlemolecular composition. A monoclonal antibody composition displays asingle binding specificity and affinity for a particular epitope. Amonoclonal antibody can be made by hybridoma technology or by methodsthat do not use hybridoma technology (e.g., recombinant methods).

An “effectively human” protein is a protein that does not evoke aneutralizing antibody response, e.g., the human anti-murine antibody(HAMA) response. HAMA can be problematic in a number of circumstances,e.g., if the antibody molecule is administered repeatedly, e.g., intreatment of a chronic or recurrent disease condition. A HAMA responsecan make repeated antibody administration potentially ineffectivebecause of an increased antibody clearance from the serum (see, e.g.,Saleh et al., Cancer Immunol. Immunother., 32:180-190 (1990)) and alsobecause of potential allergic reactions (see, e.g., LoBuglio et al.,Hybridoma, 5:5117-5123 (1986)).

The antibody molecule can be a polyclonal or a monoclonal antibody. Insome embodiments, the antibody can be recombinantly produced, e.g.,produced by any suitable phage display or combinatorial methods.

Various phage display and combinatorial methods for generatingantibodies are known in the art (as described in, e.g., Ladner et al.U.S. Pat. No. 5,223,409; Kang et al. International Publication No WO92/18619; Dower et al. International Publication No. WO 91/17271; Winteret al. International Publication WO 92/20791; Markland et al.International Publication No. WO 92/15679; Breitling et al.International Publication WO 93/01288; McCafferty et al. InternationalPublication No. WO 92/01047; Garrard et al. International PublicationNo. WO 92/09690; Ladner et al. International Publication No. WO90/02809; Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et al.(1992) Hum Antibod Hybridomas 3:81-85; Huse et al. (1989) Science246:1275-1281; Griffths et al. (1993) EMBO J 12:725-734; Hawkins et al.(1992) J Mol Biol 226:889-896; Clackson et al. (1991) Nature352:624-628; Gram et al. (1992) PNAS 89:3576-3580; Garrad et al. (1991)Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res19:4133-4137; and Barbas et al. (1991) PNAS 88:7978-7982, the contentsof all of which are incorporated by reference herein).

In some embodiments, the antibody is a fully human antibody (e.g., anantibody made in a mouse which has been genetically engineered toproduce an antibody from a human immunoglobulin sequence), or anon-human antibody, e.g., a rodent (mouse or rat), goat, primate (e.g.,monkey), camel antibody. In certain embodiments, the non-human antibodyis a rodent (mouse or rat antibody). Methods of producing rodentantibodies are known in the art.

Human monoclonal antibodies can be generated using transgenic micecarrying the human immunoglobulin genes rather than the mouse system.Splenocytes from these transgenic mice immunized with the antigen ofinterest are used to produce hybridomas that secrete human mAbs withspecific affinities for epitopes from a human protein (see, e.g., Woodet al. International Application WO 91/00906, Kucherlapati et al. PCTpublication WO 91/10741; Lonberg et al. International Application WO92/03918; Kay et al. International Application 92/03917; Lonberg, N. etal. 1994 Nature 368:856-859; Green, L. L. et al. 1994 Nature Genet.7:13-21; Morrison, S. L. et al. 1994 Proc. Natl. Acad. Sci. USA81:6851-6855; Bruggeman et al. 1993 Year Immunol 7:33-40; Tuaillon etal. 1993 PNAS 90:3720-3724; Bniggeman et al. 1991 Eur J Immunol21:1323-1326).

An antibody can be one in which the variable region, or a portionthereof, e.g., the CDRs, are generated in a non-human organism, e.g., arat or mouse. Chimeric, CDR-grafted, and humanized antibodies are alsocontemplated. Antibodies generated in a non-human organism, e.g., a rator mouse, and then modified, e.g., in the variable framework or constantregion, to decrease antigenicity in a human are also contemplated.

Chimeric antibodies can be produced by any suitable recombinant DNAtechnique. Several are known in the art (see Robinson et al.,International Patent Publication PCT/US86/02269; Akira, et al., EuropeanPatent Application 184,187; Taniguchi, M., European Patent Application171,496; Morrison et al., European Patent Application 173,494; Neubergeret al., International Application WO 86/01533; Cabilly et al. U.S. Pat.No. 4,816,567; Cabilly et al., European Patent Application 125,023;Better et al. (1988 Science 240:1041-1043); Liu et al. (1987) PNAS84:3439-3443; Liu et al., 1987, J. Immunol. 139:3521-3526; Sun et al.(1987) PNAS 84:214-218; Nishimura et al., 1987, Canc. Res. 47:999-1005;Wood et al. (1985) Nature 314:446-449; and Shaw et al., 1988, J. NatlCancer Inst. 80:1553-1559).

A humanized or CDR-grafted antibody will have at least one or two butgenerally all three recipient CDRs (of heavy and or light immunoglobulinchains) replaced with a donor CDR. The antibody may be replaced with atleast a portion of a non-human CDR or only some of the CDRs may bereplaced with non-human CDRs. It is only necessary to replace the numberof CDRs required for binding of the humanized antibody to EDIII. In someembodiments, the donor will be a rodent antibody, e.g., a rat or mouseantibody, and the recipient will be a human framework or a humanconsensus framework. Typically, the immunoglobulin providing the CDRs iscalled the “donor” and the immunoglobulin providing the framework iscalled the “acceptor.” In some embodiments, the donor immunoglobulin isa non-human (e.g., rodent). The acceptor framework is typically anaturally-occurring (e.g., a human) framework or a consensus framework,or a sequence about 85% or higher, e.g., 90%, 95%, 99% or higheridentical thereto.

As used herein, the term “consensus sequence” refers to the sequenceformed from the most frequently occurring amino acids (or nucleotides)in a family of related sequences (See e.g., Winnaker, From Genes toClones (Verlagsgesellschaft, Weinheim, Germany 1987). In a family ofproteins, each position in the consensus sequence is occupied by theamino acid occurring most frequently at that position in the family. Iftwo amino acids occur equally frequently, either can be included in theconsensus sequence. A “consensus framework” refers to the frameworkregion in the consensus immunoglobulin sequence.

An antibody can be humanized by any suitable method, and several suchmethods known in the art (see e.g., Morrison, S. L., 1985, Science229:1202-1207, by Oi et al., 1986, BioTechniques 4:214, and by Queen etal. U.S. Pat. No. 5,585,089, U.S. Pat. No. 5,693,761 and U.S. Pat. No.5,693,762, the contents of all of which are hereby incorporated byreference).

Humanized or CDR-grafted antibodies can be produced by CDR-grafting orCDR substitution, wherein one, two, or all CDRs of an immunoglobulinchain can be replaced. See e.g., U.S. Pat. No. 5,225,539; Jones et al.1986 Nature 321:552-525; Verhoeyan et al. 1988 Science 239:1534; Beidleret al. 1988 J. Immunol. 141:4053-4060; Winter U.S. Pat. No. 5,225,539,the contents of all of which are hereby expressly incorporated byreference. Winter describes a CDR-grafting method which may be used toprepare humanized antibodies (UK Patent Application GB 2188638A, filedon Mar. 26, 1987; Winter U.S. Pat. No. 5,225,539), the contents of whichis expressly incorporated by reference.

Also provided are humanized antibodies in which specific amino acidshave been substituted, deleted or added. Criteria for selecting aminoacids from the donor are described in, e.g., U.S. Pat. No. 5,585,089,e.g., columns 12-16 of U.S. Pat. No. 5,585,089, the contents of whichare hereby incorporated by reference. Other techniques for humanizingantibodies are described in Padlan et al. EP 519596 A1, published onDec. 23, 1992.

The antibody molecule can be a single chain antibody. A single-chainantibody (scFV) may be engineered (see, for example, Colcher, D. et al.(1999) Ann NY Acad Sci 880:263-80; and Reiter, Y. (1996) Clin Cancer.Res 2:245-52). The single chain antibody can be dimerized ormultimerized to generate multivalent antibodies having specificities fordifferent epitopes of the same target protein.

In some embodiments, the antibody molecule has a heavy chain constantregion chosen from, e.g., the heavy chain constant regions of IgG1,IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE; particularly, chosenfrom, e.g., the (e.g., human) heavy chain constant regions of IgG1,IgG2, IgG3, and IgG4. In another embodiment, the antibody molecule has alight chain constant region chosen from, e.g., the (e.g., human) lightchain constant regions of kappa or lambda. The constant region can bealtered, e.g., mutated, to modify the properties of the antibody (e.g.,to increase or decrease one or more of: Fc receptor binding, antibodyglycosylation, the number of cysteine residues, effector cell function,and/or complement function). In some embodiments the antibody haseffector function and can fix complement. In other embodiments theantibody does not recruit effector cells or fix complement. In certainembodiments, the antibody has reduced or no ability to bind an Fcreceptor. For example, it may be an isotype or subtype, fragment orother mutant, which does not support binding to an Fc receptor, e.g., ithas a mutagenized or deleted Fc receptor binding region.

The antibody constant region is altered in some embodiments. Methods foraltering an antibody constant region are known in the art. Antibodieswith altered function, e.g. altered affinity for an effector ligand,such as FcR on a cell, or the C1 component of complement can be producedby replacing at least one amino acid residue in the constant portion ofthe antibody with a different residue (see e.g., EP 388,151 A1, U.S.Pat. No. 5,624,821 and U.S. Pat. No. 5,648,260, the contents of all ofwhich are hereby incorporated by reference). Amino acid mutations whichstabilize antibody structure, such as S228P (EU nomenclature, S241P inKabat nomenclature) in human IgG4 are also contemplated. Similar type ofalterations could be described which if applied to the murine, or otherspecies immunoglobulin would reduce or eliminate these functions.

In some embodiments, the only amino acids in the anti-dengue antibodymolecule are canonical amino acids. In some embodiments, the anti-dengueantibody molecule comprises naturally-occurring amino acids; analogs,derivatives and congeners thereof; amino acid analogs having variantside chains; and/or all stereoisomers of any of any of the foregoing.The anti-dengue antibody molecule may comprise the D- or L-opticalisomers of amino acids and peptidomimetics.

A polypeptide of an anti-dengue antibody molecule may be linear orbranched, it may comprise modified amino acids, and it may beinterrupted by non-amino acids. The antibody molecule may also bemodified; for example, by disulfide bond formation, glycosylation,lipidation, acetylation, phosphorylation, or any other manipulation,such as conjugation with a labeling component. The polypeptide can beisolated from natural sources, can be a produced by recombinanttechniques from a eukaryotic or prokaryotic host, or can be a product ofsynthetic procedures.

The anti-dengue antibody molecule can be used alone in unconjugatedform, or can be bound to a substance, e.g., a toxin or moiety (e.g., atherapeutic drug; a compound emitting radiation; molecules of plant,fungal, or bacterial origin; or a biological protein (e.g., a proteintoxin) or particle (e.g., a recombinant viral particle, e.g., via aviral coat protein). For example, the anti-dengue antibody can becoupled to a radioactive isotope such as an α-, β-, or γ-emitter, or aβ- and γ-emitter.

An antibody molecule can be derivatized or linked to another functionalmolecule (e.g., another peptide or protein). As used herein, a“derivatized” antibody molecule is one that has been modified. Methodsof derivatization include but are not limited to the addition of afluorescent moiety, a radionucleotide, a toxin, an enzyme or an affinityligand such as biotin. Accordingly, the antibody molecules are intendedto include derivatized and otherwise modified forms of the antibodiesdescribed herein, including immunoadhesion molecules. For example, anantibody molecule can be functionally linked (by chemical coupling,genetic fusion, noncovalent association or otherwise) to one or moreother molecular entities, such as another antibody (e.g., a bispecificantibody or a diabody), a detectable agent, a toxin, a pharmaceuticalagent, and/or a protein or peptide that can mediate association of theantibody or antibody portion with another molecule (such as astreptavidin core region or a polyhistidine tag).

Some types of derivatized antibody molecule are produced by crosslinkingtwo or more antibodies (of the same type or of different types, e.g., tocreate bispecific antibodies). Suitable crosslinkers include those thatare heterobifunctional, having two distinctly reactive groups separatedby an appropriate spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimideester) or homobifunctional (e.g., disuccinimidyl suberate). Such linkersare available from Pierce Chemical Company, Rockford, Ill.

Useful detectable agents with which an anti-dengue antibody molecule maybe derivatized (or labeled) to include fluorescent compounds, variousenzymes, prosthetic groups, luminescent materials, bioluminescentmaterials, fluorescent emitting metal atoms, e.g., europium (Eu), andother anthanides, and radioactive materials (described below). Exemplaryfluorescent detectable agents include fluorescein, fluoresceinisothiocyanate, rhodamine, 5dimethylamine-1-napthalenesulfonyl chloride,phycoerythrin and the like. An antibody may also be derivatized withdetectable enzymes, such as alkaline phosphatase, horseradishperoxidase, β-galactosidase, acetylcholinesterase, glucose oxidase andthe like. When an antibody is derivatized with a detectable enzyme, itis detected by adding additional reagents that the enzyme uses toproduce a detectable reaction product. For example, when the detectableagent horseradish peroxidase is present, the addition of hydrogenperoxide and diaminobenzidine leads to a colored reaction product, whichis detectable. An antibody molecule may also be derivatized with aprosthetic group (e.g., streptavidin/biotin and avidin/biotin). Forexample, an antibody may be derivatized with biotin, and detectedthrough indirect measurement of avidin or streptavidin binding. Examplesof suitable fluorescent materials include umbelliferone, fluorescein,fluorescein isothiocyanate, rhodamine, dichlorotriazinylaminefluorescein, dansyl chloride or phycoerythrin; an example of aluminescent material includes luminol; and examples of bioluminescentmaterials include luciferase, luciferin, and aequorin.

Labeled antibody molecule can be used, for example, diagnosticallyand/or experimentally in a number of contexts, including (i) to isolatea predetermined antigen by standard techniques, such as affinitychromatography or immunoprecipitation; (ii) to detect a predeterminedantigen (e.g., in a cellular lysate or cell supernatant) in order toevaluate the abundance and pattern of expression of the protein; (iii)to monitor protein levels in tissue as part of a clinical testingprocedure, e.g., to determine the efficacy of a given treatment regimen.

An antibody molecule may be conjugated to another molecular entity,typically a label or a therapeutic (e.g., inununomodulatory,immunostimularoty, cytotoxic, or cytostatic) agent or moiety.Radioactive isotopes can be used in diagnostic or therapeuticapplications. Radioactive isotopes that can be coupled to theanti-dengue antibodies include, but are not limited to α-, β-, orγ-emitters, or β -and γ-emitters. Such radioactive isotopes include, butare not limited to iodine (¹³¹I or ¹²⁵I), yttrium (⁹⁰Y), lutetium(¹⁷⁷Lu), actinium (²²⁵Ac), praseodymium, astatine (²¹¹At), rhenium(¹⁸⁶Re), bismuth (²¹²Bi or ²¹³Bi) indium (¹¹¹In), technetium (⁹⁹mTc),phosphorus (³²P), rhodium (¹⁸⁸Rh), sulfur (³⁵S), carbon (¹⁴C), tritium(³H), chromium (⁵¹Cr), chlorine (³⁶Cl), cobalt (⁵⁷Co or ⁵⁸Co), iron(⁵⁹Fe), selenium (⁷⁵Se), or gallium (⁶⁷Ga). Radioisotopes useful astherapeutic agents include yttrium (⁹⁰Y), lutetium (¹⁷⁷Lu), actinium(²²⁵Ac), praseodymium, astatine (²¹¹At), rhenium (¹⁸⁶Re), bismuth (²¹²Bior ²¹³Bi), and rhodium (¹⁸⁸Rh). Radioisotopes useful as labels, e.g.,for use in diagnostics, include iodine (¹³¹I or ¹²⁵I), indium (¹¹¹In),technetium (⁹⁹mTc), phosphorus (³²P), carbon (¹⁴C), and tritium (³H), orone or more of the therapeutic isotopes listed above.

The present disclosure provides radiolabeled antibody molecules andmethods of labeling the same. In some embodiments, a method of labelingan antibody molecule is disclosed. The method includes contacting anantibody molecule, with a chelating agent, to thereby produce aconjugated antibody. The conjugated antibody is radiolabeled with aradioisotope, e.g., ¹¹¹Indium, ⁹⁰Yttrium and ¹⁷⁷Lutetium, to therebyproduce a labeled antibody molecule.

As is discussed above, the antibody molecule can be conjugated to atherapeutic agent. Therapeutically active radioisotopes have alreadybeen mentioned. Examples of other therapeutic agents include anti-viralagents.

In some aspects, this disclosure provides a method of providing anantibody molecule disclosed herein. The method includes: providing anantigen, e.g., a dengue virus E protein or portion thereof; obtaining anantibody molecule that specifically binds to the antigen; evaluatingefficacy of the antibody molecule in modulating activity of the antigenand/or organism expressing the antigen, e.g., dengue virus. The methodcan further include administering the antibody molecule, including aderivative thereof (e.g., a humanized antibody molecule) to a subject,e.g., a human.

This disclosure provides an isolated nucleic acid molecule encoding theabove antibody molecule, vectors and host cells thereof. The nucleicacid molecule includes but is not limited to RNA, genomic DNA and cDNA.

Animal Models

The antibody molecules described herein can be evaluated in an animalmodel. For example, an animal model can be used to test the efficacy ofan antibody molecule described herein in reducing dengue viralinfection, replication and/or transmission. Exemplary animal models thatcan be used for evaluating an antibody molecule described hereininclude, but are not limited to, AG129 mouse models (e.g., as describedin Tharakaraman et al., Proc Natl Acad Sci USA. 2013; 110(17):E1555-64;Johnson et al. J Virol. 1999; 73(1):783-6); non-mouse adapted mousemodels (e.g., non-mouse adapted DENV-2 D2Y98P mouse model as describedin Tan et al. PLoS Negl Trop Dis. 2010; 4(4):e672); humanized mousemodels (e.g., as described in Sridharan et al. J Virol. 2013;87(21):11648-58); non-human primate models (e.g., as described inGoncalvez et al. Proc Natl Acad Sci USA. 2007; 104(22):9422-7); andmosquito models (e.g., as described in Vu et al. PLoS Negl Trop Dis.2010; 4(7):e757).

The AG129 mouse strain, which lacks both type-I and type-II interferonreceptors, is an animal model that replicates certain diseasemanifestations observed in clinical cases of dengue, including viremiaand other signs of disease (Tharakaraman et al., Proc Natl Acad Sci USA.2013; 110(17):E1555-64; Johnson et al. J Virol. 1999; 73(1):783-6). Thismodel is useful in evaluation of antiviral treatments and can also beused in proof of principle studies. Briefly, the AG129 (which isdeficient in IFN-γ/β and IFN-γreceptors) mouse is challenged with denguevirus, and a candidate therapeutic antibody molecule is administered.Typically, viremia (virus titer in a blood sample) is the endpoint ofthe experiment. Viremia can be measured, e.g., with quantitative RT-PCR.An exemplary AG129 mouse model is described in Example 10.

Non-mouse adapted mouse models can be generated, e.g., using a non-mouseadapted DEN2 virus strain (D2Y98P) that is highly infectious in AG129mice upon intraperitoneal administration (Tan et al. PLoS Negl Trop Dis.2010; 4(4):e672). Infection with a high dose of D2Y98P can inducecytokine storm, massive organ damage, and severe vascular leakage,leading to haemorrhage and rapid death of the animals at the peak ofviremia. Infection with a low dose of D2Y98P can lead to asymptomaticviral dissemination and replication in relevant organs, followed bynon-paralytic death of the animals few days after virus clearance,similar to the disease kinetic in humans. Spleen damage, liverdysfunction and increased vascular permeability, but no hemorrhage, canbe observed in moribund animals, suggesting intact vascular integrity, acardinal feature in dengue shock syndrome.

Humanized mouse models can be generated, e.g., by adoptive transfer ofhuman CD34⁺ fetal liver cells into NOD-scid 112rg^(−/−) (NSG) mice thatdevelop significant levels of human platelets, monocytes/macrophages,and hepatocytes (Sridharan et al. J Virol. 2013; 87(21):11648-58).Infection of these mice with dengue virus such as DENV serotype 2(DENV-2) can recapture certain characteristic features of dengue viralinfection in humans, e.g., transient leukopenia and thrombocytopenia.

Non-human primate models can be generated, e.g., in juvenile rhesusmonkeys after DENV challenge, as described in Goncalvez et al. Proc NatlAcad Sci USA. 2007; 104(22):9422-7. The viremia titers of infectedmonkeys can be determined, e.g., by quantitative PCR or Focus FormingUnits (FFU) assay.

Mosquito models can also be used to evaluate inhibitory activity ofantibodies against dengue virus, e.g., neutralization of viral infectionor reduction of transmission between infected subjects and mosquitoes.Dengue virus is a mosquito transmitted RNA virus. Certain dengue viruscan develop in vivo fitness advantage, which may result in higherprobability of human-to-mosquito transmission (Vu et al., PLoS Negl TropDis. 2010; 4(7):e757). To establish a mosquito model, blood containingvirus and antibody can be fed to mosquitoes. Viral load in mosquitoes'abdomens can be measured by qRT-PCR. An exemplary mosquito model isdescribed in Example 13.

Pharmaceutical Compositions and Kits

In some aspects, this disclosure provides compositions, e.g.,pharmaceutically acceptable compositions, which include an anti-dengueantibody molecule described herein, formulated together with apharmaceutically acceptable carrier. As used herein, “pharmaceuticallyacceptable carrier” includes any and all solvents, dispersion media,isotonic and absorption delaying agents, and the like that arephysiologically compatible. The carrier can be suitable for intravenous,intramuscular, subcutaneous, parenteral, rectal, spinal or epidermaladministration (e.g., by injection or infusion). In certain embodiments,less than about 5%, e.g., less than about 4%, 3%, 2%, or 1% of theantibody molecules in the pharmaceutical composition are present asaggregates. In other embodiments, at least about 95%, e.g., at leastabout 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.8%, or more of the antibodymolecules in the pharmaceutical composition are present as monomers. Insome embodiments, the level of antibody aggregates or monomers isdetermined by chromatography, e.g., high performance size exclusionchromatography (HP-SEC).

The compositions set out herein may be in a variety of forms. Theseinclude, for example, liquid, semi-solid and solid dosage forms, such asliquid solutions (e.g., injectable and infusible solutions), dispersionsor suspensions, liposomes, and suppositories. A suitable form depends onthe intended mode of administration and therapeutic application. Typicalsuitable compositions are in the form of injectable or infusiblesolutions. One suitable mode of administration is parenteral (e.g.,intravenous, subcutaneous, intraperitoneal, intramuscular). In someembodiments, the antibody molecule is administered by intravenousinfusion or injection. In certain embodiments, the antibody isadministered by intramuscular or subcutaneous injection.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal, epidural and intrasternal injection andinfusion.

Therapeutic compositions typically should be sterile and stable underthe conditions of manufacture and storage. The composition can beformulated as a solution, microemulsion, dispersion, liposome, or otherordered structure suitable to high antibody concentration. Sterileinjectable solutions can be prepared by incorporating the activecompound (i.e., antibody or antibody portion) in the required amount inan appropriate solvent with one or a combination of ingredientsenumerated above, as required, followed by filtered sterilization.Generally, dispersions are prepared by incorporating the active compoundinto a sterile vehicle that contains a basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofsterile powders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and freeze-dryingthat yields a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.The proper fluidity of a solution can be maintained, for example, by theuse of a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prolonged absorption of injectable compositions can be brought about byincluding in the composition an agent that delays absorption, forexample, monostearate salts and gelatin.

The antibody molecules can be administered by a variety of methods.Several are known in the art, and for many therapeutic applications, anappropriate route/mode of administration is intravenous injection orinfusion. For example, the antibody molecules can be administered byintravenous infusion at a rate of less than 10 mg/min; preferably lessthan or equal to 5 mg/min to reach a dose of about 1 to 100 mg/m²,preferably about 5 to 50 mg/m², about 7 to 25 mg/m² and more preferably,about 10 mg/m². As will be appreciated by the skilled artisan, the routeand/or mode of administration will vary depending upon the desiredresults. In certain embodiments, the active compound may be preparedwith a carrier that will protect the compound against rapid release,such as a controlled release formulation, including implants,transdermal patches, and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Many methods for the preparationof such formulations are patented or generally known to those skilled inthe art. See, e.g., Sustained and Controlled Release Drug DeliverySystems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

In certain embodiments, an antibody molecule can be orally administered,for example, with an inert diluent or an assimilable edible carrier. Theantibody molecule (and other ingredients, if desired) may also beenclosed in a hard or soft shell gelatin capsule, compressed intotablets, or incorporated directly into the subject's diet. For oraltherapeutic administration, the antibody molecule may be incorporatedwith excipients and used in the form of ingestible tablets, buccaltablets, troches, capsules, elixirs, suspensions, syrups, wafers, andthe like. To administer an antibody molecule by other than parenteraladministration, it may be necessary to coat compound with, orco-administer the compound with, a material to prevent its inactivation.Therapeutic compositions can also be administered with medical devices,and several are known in the art.

Dosage regimens are adjusted to provide the desired response (e.g., atherapeutic response). For example, a single bolus may be administered,several divided doses may be administered over time or the dose may beproportionally reduced or increased as indicated by the exigencies ofthe therapeutic situation. It is especially advantageous to formulateparenteral compositions in dosage unit form for ease of administrationand uniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subjects tobe treated; each unit contains a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms are dictated by and directly dependent on (a)the unique characteristics of the antibody molecule and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of compounding such an antibody molecule for the treatment ofsensitivity in individuals.

An exemplary, non-limiting range for a therapeutically orprophylactically effective amount of an antibody molecule is 0.1-20mg/kg, more preferably 1-10 mg/kg. The antibody molecule can beadministered by intravenous infusion at a rate of less than 10 mg/min,preferably less than or equal to 5 mg/min to reach a dose of about 1 to100 mg/m², preferably about 5 to 50 mg/m², about 7 to 25 mg/m², and morepreferably, about 10 mg/m². It is to be noted that dosage values mayvary with the type and severity of the condition to be alleviated. It isto be further understood that for any particular subject, specificdosage regimens should be adjusted over time according to the individualneed and the professional judgment of the person administering orsupervising the administration of the compositions, and that dosageranges set forth herein are exemplary only and are not intended to limitthe scope or practice of the claimed compositions.

The pharmaceutical compositions herein may include a “therapeuticallyeffective amount” or a “prophylactically effective amount” of anantibody molecule. A “therapeutically effective amount” refers to anamount effective, at dosages and for periods of time necessary, toachieve the desired therapeutic result. A therapeutically effectiveamount of the modified antibody or antibody fragment may vary accordingto factors such as the disease state, age, sex, and weight of theindividual, and the ability of the antibody or antibody portion toelicit a desired response in the individual. A therapeutically effectiveamount is also one in which any toxic or detrimental effects of theantibody molecule is outweighed by the therapeutically beneficialeffects. A “therapeutically effective dosage” preferably inhibits ameasurable parameter by at least about 20%, more preferably by at leastabout 40%, even more preferably by at least about 60%, and still morepreferably by at least about 80% relative to untreated subjects. Themeasurable parameter may be, e.g., viral load, fever, headache, muscleor joint pains, skin rash, bleeding, reduced platelet levels, andreduced blood pressure. The ability of an antibody molecule to inhibit ameasurable parameter can be evaluated in an animal model systempredictive of efficacy in dengue fever. Alternatively, this property ofa composition can be evaluated by examining the ability of the antibodymolecule to neutralize dengue virus, e.g., by assaying focus formationin vitro.

A “prophylactically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredprophylactic result. Typically, since a prophylactic dose is used insubjects prior to or at an earlier stage of disease, theprophylactically effective amount will be less than the therapeuticallyeffective amount.

Also within this disclosure is a kit comprising an antibody moleculedescribed herein. The kit can include one or more other elementsincluding: instructions for use; other reagents, e.g., a label, atherapeutic agent, or an agent useful for chelating, or otherwisecoupling, an antibody to a label or therapeutic agent, or aradioprotective composition; devices or other materials for preparingthe antibody molecule for administration; pharmaceutically acceptablecarriers; and devices or other materials for administration to asubject.

Nucleic Acids

The present disclosure also features nucleic acids comprising nucleotidesequences that encode heavy and light chain variable regions and CDRs ofthe anti-dengue antibody molecules, as described herein. For example,the present disclosure features a first and second nucleic acid encodingheavy and light chain variable regions, respectively, of an anti-dengueantibody molecule chosen from one or more of the antibody moleculesdisclosed herein, e.g., an antibody of Table 1, or a portion of anantibody, e.g., the variable regions of Table 2. The nucleic acid cancomprise a nucleotide sequence encoding any one of the amino acidsequences in the tables herein, or a sequence substantially identicalthereto (e.g., a sequence at least about 85%, 90%, 95%, 99% or moreidentical thereto, or which differs by no more than 3, 6, 15, 30, or 45nucleotides from the sequences shown in the tables herein.

In certain embodiments, the nucleic acid can comprise a nucleotidesequence encoding at least one, two, or three CDRs from a heavy chainvariable region having an amino acid sequence as set forth in the tablesherein, or a sequence substantially homologous thereto (e.g., a sequenceat least about 85%, 90%, 95%, 99% or more identical thereto, and/orhaving one or more substitutions, e.g., conserved substitutions). Insome embodiments, the nucleic acid can comprise a nucleotide sequenceencoding at least one, two, or three CDRs from a light chain variableregion having an amino acid sequence as set forth in the tables herein,or a sequence substantially homologous thereto (e.g., a sequence atleast about 85%, 90%, 95%, 99% or more identical thereto, and/or havingone or more substitutions, e.g., conserved substitutions). In someembodiments, the nucleic acid can comprise a nucleotide sequenceencoding at least one, two, three, four, five, or six CDRs from heavyand light chain variable regions having an amino acid sequence as setforth in the tables herein, or a sequence substantially homologousthereto (e.g., a sequence at least about 85%, 90%, 95%, 99% or moreidentical thereto, and/or having one or more substitutions, e.g.,conserved substitutions).

In certain embodiments, the nucleic acid can comprise a nucleotidesequence encoding at least one, two, or three CDRs from a heavy chainvariable region having the nucleotide sequence as set forth in Table 5herein, a sequence substantially homologous thereto (e.g., a sequence atleast about 85%, 90%, 95%, 99% or more identical thereto, and/or capableof hybridizing under the stringency conditions described herein). Insome embodiments, the nucleic acid can comprise a nucleotide sequenceencoding at least one, two, or three CDRs from a light chain variableregion having the nucleotide sequence as set forth in Table 5 herein, ora sequence substantially homologous thereto (e.g., a sequence at leastabout 85%, 90%, 95%, 99% or more identical thereto, and/or capable ofhybridizing under the stringency conditions described herein). Incertain embodiments, the nucleic acid can comprise a nucleotide sequenceencoding at least one, two, three, four, five, or six CDRs from heavyand light chain variable regions having the nucleotide sequence as setforth in Table 5 herein, or a sequence substantially homologous thereto(e.g., a sequence at least about 85%, 90%, 95%, 99% or more identicalthereto, and/or capable of hybridizing under the stringency conditionsdescribed herein).

In certain embodiments, the nucleic acid comprises a nucleotide sequenceas set forth in Table 5 herein or a sequence substantially homologousthereto (e.g., a sequence at least about 85%, 90%, 95%, 99% or moreidentical thereto, and/or capable of hybridizing under the stringencyconditions described herein). In some embodiments, the nucleic acidcomprises a portion of a nucleotide sequence as set forth_(—) in Table 5herein or a sequence substantially homologous thereto (e.g., a sequenceat least about 85%, 90%, 95%, 99% or more identical thereto, and/orcapable of hybridizing under the stringency conditions describedherein). The portion may encode, for example, a variable region (e.g.,VH or VL); one, two, or three or more CDRs; or one, two, three, or fouror more framework regions.

The nucleic acids disclosed herein include deoxyribonucleotides orribonucleotides, or analogs thereof. The polynucleotide may be eithersingle-stranded or double-stranded, and if single-stranded may be thecoding strand or non-coding (antisense) strand. A polynucleotide maycomprise modified nucleotides, such as methylated nucleotides andnucleotide analogs. The sequence of nucleotides may be interrupted bynon-nucleotide components. A polynucleotide may be further modifiedafter polymerization, such as by conjugation with a labeling component.The nucleic acid may be a recombinant polynucleotide, or apolynucleotide of genomic, cDNA, semisynthetic, or synthetic originwhich either does not occur in nature or is linked to anotherpolynucleotide in a nonnatural arrangement.

In some aspects, the application features host cells and vectorscontaining the nucleic acids described herein. The nucleic acids may bepresent in a single vector or separate vectors present in the same hostcell or separate host cell, as described in more detail hereinbelow.

Vectors

Further provided herein are vectors comprising nucleotide sequencesencoding an antibody molecule described herein. In some embodiments, thevectors comprise nucleotides encoding an antibody molecule describedherein. In some embodiments, the vectors comprise the nucleotidesequences described herein. The vectors include, but are not limited to,a virus, plasmid, cosmid, lambda phage or a yeast artificial chromosome(YAC).

Numerous vector systems can be employed. For example, one class ofvectors utilizes DNA elements which are derived from animal viruses suchas, for example, bovine papilloma virus, polyoma virus, adenovirus,vaccinia virus, baculovirus, retroviruses (Rous Sarcoma Virus, MMTV orMOMLV) or SV40 virus. Another class of vectors utilizes RNA elementsderived from RNA viruses such as Semliki Forest virus, Eastern EquineEncephalitis virus and Flaviviruses.

Additionally, cells which have stably integrated the DNA into theirchromosomes may be selected by introducing one or more markers whichallow for the selection of transfected host. cells. The marker mayprovide, for example, prototropy to an auxotrophic host, biocideresistance, (e.g., antibiotics), or resistance to heavy metals such ascopper, or the like. The selectable marker gene can be either directlylinked to the DNA sequences to be expressed, or introduced into the samecell by cotransformation. Additional elements may also be needed foroptimal synthesis of mRNA. These elements may include splice signals, aswell as transcriptional promoters, enhancers, and termination signals.

Once the expression vector or DNA sequence containing the constructs hasbeen prepared for expression, the expression vectors may be transfectedor introduced into an appropriate host cell. Various techniques may beemployed to achieve this, such as, for example, protoplast fusion,calcium phosphate precipitation, electroporation, retroviraltransduction, viral transfection, gene gun, lipid based transfection orother conventional techniques. In the case of protoplast fusion, thecells are grown in media and screened for the appropriate activity.

Methods and conditions for culturing the resulting transfected cells andfor recovering the antibody molecule produced are known to those skilledin the art, and may be varied or optimized depending upon the specificexpression vector and mammalian host cell employed, based upon thepresent description.

Cells

The present disclosure also provides host cells comprising a nucleicacid encoding an antibody molecule as described herein. For example, thehost cells may comprise a nucleic acid of Table 5, a sequencesubstantially homologous thereto (e.g., a sequence at least about 85%,90%, 95%, 99% or more identical thereto, and/or capable of hybridizingunder the stringency conditions described herein), or a portion of oneof said nucleic acids. Additionally, the host cells may comprise anucleic acid encoding an amino acid sequence of Table 2 or Table 3, asequence substantially homologous thereto (e.g., a sequence at leastabout 85%, 90%, 95%, 99% or more identical thereto), or a portion of oneof said sequences.

In some embodiments, the host cells are genetically engineered tocomprise nucleic acids encoding the antibody_molecule.

In certain embodiments, the host cells are genetically engineered byusing an expression cassette. The phrase “expression cassette,” refersto nucleotide sequences, which are capable of affecting expression of agene in hosts compatible with such sequences. Such cassettes may includea promoter, an open reading frame with or without introns, and atermination signal. Additional factors necessary or helpful in effectingexpression may also be used, such as, for example, an induciblepromoter.

The disclosure also provides host cells comprising the vectors describedherein.

The cell can be, but is not limited to, a eukaryotic cell, a bacterialcell, an insect cell, or a human cell. Suitable eukaryotic cellsinclude, but are not limited to, Vero cells, HeLa cells, COS cells, CHOcells, HEK293 cells, BHK cells and MDCKII cells. Suitable insect cellsinclude, but are not limited to, Sf9 cells.

Uses of Anti-Dengue Antibody Molecules

The antibody molecules disclosed herein have in vitro and in vivodiagnostic, as well as therapeutic and prophylactic utilities. In someembodiments, the antibody molecules neutralize dengue virus. Forexample, these molecules can be administered to cells in culture, invitro or ex vivo, or to a subject, e.g., a human subject, e.g., in vivo,to neutralize dengue virus. Accordingly, in some aspects, the disclosureprovides a method of treating a dengue virus infection in a subject,comprising administering to the subject an antibody molecule describedherein, such that the dengue virus infection is treated. For example,these antibody molecules can be administered to cells in culture, e.g.in vitro or ex vivo, or in a subject, e.g., in vivo, to treat, prevent,and/or diagnose a dengue virus infection.

As used herein, the term “subject” is intended to include human andnon-human animals. In some embodiments, the subject is a human subject,e.g., a human patient infected with dengue virus or at risk of beinginfected with dengue virus. The term “non-human animals” includesmammals and non-mammals, such as non-human primates. In someembodiments, the subject is a human. The methods and compositionsdescribed herein are suitable for treating human patients infected withdengue virus. Patients infected with dengue virus include those who havebeen exposed to the virus but are (at least temporarily) asymptomatic,patients having dengue fever, patients having dengue hemorrhagic fever,and patients having dengue shock syndrome.

Methods of Treating Dengue Virus

Dengue virus displays an E (envelope) protein on the viral surface. TheE protein contributes to the attachment of the virus to a host cell. TheE protein comprises a DI domain (a nine-stranded beta-barrel) a DIIdomain (a hydrophobic domain implicated in fusion with the host cell),and a DIII domain (an extracellular domain). While not wishing to bebound by theory, in some embodiments, the antibody molecules describedherein can neutralize dengue virus by binding to its E protein DIII(EDIII) domain, e.g., by preventing the virus from fusing with a hostcell, preventing the virus from binding to a host cell, disrupting thestructure of the E protein, or destabilizing the virus.

Dengue fever is an infectious disease, usually mosquito-borne, caused bythe dengue virus. The initial infection is often followed by a briefasymptomatic period, usually 4-7 days. Sometimes an infected patientdoes not develop any symptoms of dengue fever. However, in patients thatmanifest dengue fever, the characteristic symptoms are sudden-onsetfever (sometimes over 40° C.), headache, muscle and joint pains, andrash. During the febrile phase of infection, fever, pain, and headachemanifest. In some patients the febrile phase is followed by the criticalphase (associated with dengue shock syndrome and dengue hemorrhagicfever), in which patients may suffer from fluid accumulation in thechest and abdominal cavity, depletion of fluid from circulation, aninadequate supply of blood to the vital organs, and bleeding. This isfollowed by a recovery phase. In some embodiments, the antibodymolecules herein are administered to a patient in the asymptomaticperiod, the febrile phase, the critical phase, and/or the recoveryphase.

Dengue virus is typically diagnosed based on a physical exam and thepatient's reported symptoms. A probable diagnosis can be made when apatient displays a fever and at least two symptoms selected fromnausea/vomiting, rash, generalized pain, reduced white blood celllevels, or positive tourniquet test. Additional tests that indicatedengue fever include a test for reduced white blood cell count, lowplatelet levels, metabolic acidosis, elevated level of aminotransferasefrom the liver, hemoconcentration, hypoalbuminemia, detection of fluidby ultrasound (suggests dengue shock syndrome), a pulse pressure below20 mm Hg (indicates dengue shock syndrome), delayed capillary refill(indicates peripheral vascular collapse). Accordingly, in someembodiments the antibody molecules are administered to a patient thatsatisfies the aforementioned criteria.

Certain antibody molecules described herein are capable of treating atleast two, three, or four serotypes of dengue virus. Accordingly, incertain embodiments, the antibody molecule is administered to a patientinfected with or with a risk of being infected with dengue virus, whenno test has been performed to determine the serotype of the denguevirus, e.g., the serotype of the dengue virus may be unknown. In someembodiments, the dengue virus is of serotype DV-1, DV-2, DV-3, or DV-4.

The antibody molecules are typically administered at a frequency thatkeeps a therapeutically effective level of antibodies in the patient'ssystem until the patient recovers. For example, the antibody moleculesmay be administered at a frequency that achieves a serum concentrationsufficient for at least about 1, 2, 5, 10, 20, 30, or 40 antibodies tobind each virion. In some embodiments, the antibody molecules areadministered every 1, 2, 3, 4, 5, 6, or 7 days.

Methods of administering various antibody molecules are known in the artand are described below. Suitable dosages of the antibody molecules usedwill depend on the age and weight of the subject and the particular drugused.

The antibody molecules can be used by themselves or conjugated to asecond agent, e.g., an antiviral agent, toxin, or protein, e.g., asecond anti-dengue antibody. This method includes: administering theantibody molecule, alone or conjugated to a second agent, to a subjectrequiring such treatment. The antibody molecules can be used to delivera variety of therapeutic agents, e.g., a toxin or anti-viral agent, ormixtures thereof.

Combination Therapies

The anti-dengue antibody molecules can be used in combination with othertherapies. For example, the combination therapy can include ananti-dengue antibody molecule co-formulated with, and/or co-administeredwith, one or more additional therapeutic agents, e.g., anti-viral agents(including other anti-dengue antibodies), vaccines (including denguevirus vaccines), or agents that enhance an immune response. In otherembodiments, the antibody molecules are administered in combination withother therapeutic treatment modalities, such as intravenous hydration,fever-reducing agents (such as acetaminophen), or blood transfusion.Such combination therapies may advantageously utilize lower dosages ofthe administered therapeutic agents, thus avoiding possible toxicitiesor complications associated with the various monotherapies.

Administered “in combination”, as used herein, means that two (or more)different treatments are delivered to the subject before, or during thecourse of the subject's affliction with the disease. In one embodiment,two or more treatments are delivered prophylactically, e.g., before thesubject is infected or diagnosed with dengue virus. In anotherembodiment, the two or more treatments are delivered after the subjecthas been diagnosed with the dengue virus. In some embodiments, thedelivery of one treatment is still occurring when the delivery of thesecond begins, so that there is overlap. This is sometimes referred toherein as “simultaneous” or “concurrent delivery.” In other embodiments,the delivery of one treatment ends before the delivery of the othertreatment begins. In some embodiments of either case, the treatment ismore effective because of combined administration. For example, thesecond treatment is more effective, e.g., an equivalent effect is seenwith less of the second treatment, or the second treatment reducessymptoms to a greater extent, than would be seen if the second treatmentwere administered in the absence of the first treatment, or theanalogous situation is seen with the first treatment. In someembodiments, delivery is such that the reduction in a symptom, or otherparameter related to the disorder is greater than what would be observedwith one treatment delivered in the absence of the other. The effect ofthe two treatments can be partially additive, wholly additive, orgreater than additive. The delivery can be such that an effect of thefirst treatment delivered is still detectable when the second isdelivered.

The anti-viral agent may be, e.g., balapiravir, chloroquine, celgosivir,ivermectin, or Carica folia.

The vaccine may be, e.g., live, attenuated, recombinant dengue serotypes1, 2, 3, and 4 virus (e.g., clinical trial NCT01488890 by SanofiPasteur); CYD Tetravalent Dengue Vaccine (e.g., clinical trialNCT01943825, by Sanofi Pasteur), Chimeric dengue serotype (1, 2, 3, 4)(e.g., clinical trial NCT00730288 by Sanofi), CYD Dengue Vaccine (e.g.,clinical trial NCT00993447 by Sanofi), tetravalent live attenuateddengue vaccine (e.g., clinical trial NCT00322049 by GlaxoSmithKline),Tetravalent Dengue Vaccine (TVDV) (e.g., clinical trial NCT01502358 byU.S. Army Medical Research and Materiel Command), Chimeric tetravalentdengue (serotype 1, 2, 3, 4) (e.g., clinical trial NCT00842530 by SanofiPasteur), dengue lyophilized vaccine (e.g., clinical trial NCT01696422by Butantan Institute), ChimeriVax™ Tetravalent Dengue Vaccine (e.g.,clinical trial NCT00617344 by Sanofi), Bivalent CYD-1,3 Dengue (Vero)(e.g., clinical trial NCT00740155 by Sanofi Pasteur), Bivalent CYD-2,4Dengue (Vero) (e.g., clinical trial NCT00740155 by Sanofi Pasteur),Tetravalent blending VDV-2CYD-1,3,4 Dengue (Vero) (e.g., clinical trialNCT00740155 by Sanofi Pasteur), Tetravalent CYD-1,2,3,4 Dengue (Vero)(e.g., clinical trial NCT00740155 by Sanofi Pasteur), rDEN1delta30 orrDEN2/4delta30(ME) (e.g., clinical trial NCT00458120 by NationalInstitute of Allergy and Infectious Diseases), Modified Live TetravalentChimeric Dengue Vaccine (SC or ID) (e.g., clinical trial NCT01110551 byNational Institute of Allergy and Infectious Diseases), Dengue vaccine(e.g., clinical trial NCT00384670 by United States Army Medical MaterielDevelopment Activity), Investigational Vaccine for Dengue Virus Subtype2 (e.g., NCT01073306 by National Institute of Allergy and InfectiousDiseases), F17 (e.g., NCT01843621 by U.S. Army Medical Research andMateriel. Command), Post-Transfection F17 or Post-Transfection F19(e.g., clinical trial NCT00468858 by U.S. Army Medical Research andMateriel Command), DENVax (e.g., clinical trial NCT01511250 by InviragenInc.), D1ME (dengue-1 premembrane/envelope DNA vaccine) (e.g., clinicaltrial NCT00290147 by U.S. Army Office of the Surgeon General),Investigational Vaccine for DEN1 (e.g., clinical trial NCT01084291 byNational Institute of Allergy and Infectious Diseases), Live attenuatedtetravalent dengue vaccine (e.g., clinical trial NCT00350337 by WalterReed Army Institute of Research), rDEN4delta30-200,201 (e.g., clinicaltrial NCT00270699 by National Institute of Allergy and InfectiousDiseases), TetraVax-DV-TV003 or rDEN2Δ30-7169 (e.g., clinical trialNCT02021968 by National Institute of Allergy and Infectious Diseases),TetraVax-DV, optionally in admixture (e.g., clinical trial NCT01436422by National Institute of Allergy and Infectious Diseases), DEN4 VaccineCandidate (e.g., clinical trial NCT00919178 by National Institute ofAllergy and Infectious Diseases), rDEN4delta30-4995 (e.g., clinicaltrial NCT00322946 by National Institute of Allergy and InfectiousDiseases), rDEN3delta30/31-7164 (e.g., clinical trial NCT00831012 byNational Institute of Allergy and Infectious Diseases), TDENV-PIV (e.g.,clinical trial NCT01702857 by U.S. Army Medical Research and MaterielCommand), DENV-1 PIV (e.g., clinical trial NCT01502735 by U.S. ArmyMedical Research and-Materiel Command), rDEN3-3′D4delta30 (e.g.,clinical trial NCT00712803 by National Institute of Allergy andInfectious Diseases), V180 (e.g., clinical trial NCT01477580 by MerckSharp & Dohme Corp.), or DEN1-80E (e.g., clinical trial NCT00936429 byHawaii Biotech, Inc.).

The other therapy may be, for example, hypertonic sodium lactate,activated recombinant human factor VII, or anti-d (e.g., clinical trialNCT01443247 by Postgraduate Institute of Medical Education andResearch).

In certain embodiments, the additional antiviral agent is a secondanti-dengue antibody molecule, e.g., an anti-dengue antibody moleculedifferent from a first anti-dengue antibody molecule. Exemplaryanti-dengue antibody molecules that can be used in combination include,but are not limited to, any combination of the antibodies listed inTable 1 (for example, any combination of two of more of D88, F38, A48,C88, F108, B48, A68, A100, C58, C78, C68, D98, A11 (also known asmonoclonal antibody 4E5A (Tharakaraman et al., Proc Natl Acad Sci USA.2013; 110(17):E1555-64)) or B11; monoclonal antibody 4E11 (Thullier etal., J Biotechnol. 1999; 69(2-3):183-90); human antibody 14c10 (HM14c10)(Teoh et al. Sci Transl Med. 2012 Jun. 20; 4(139):139ra83); humanmonoclonal antibodies 1F4, 2D22, and 5J7 (de Alwis et al., Proc NatlAcad Sci USA. 2012; 109(19):7439-44); human monoclonal antibodies DV1.1,DV1.6, DV3.7, DV4.4, DV5.1, DV6.1, DV7.5, DV8.1, DV10.16, DV13.4,DV13.8, DV14.5, DV14.5, DV15.7, DV16.5, DV16.8, DV17.6, DV18.21, DV18.4,DV19.3, DV20.1, DV21.1, DV21.5, DV22.3, DV22.3 LALA, DV23.13, DV25.5,DV27.2, DV28.1, DV28.8, DV34.4, DV35.3, DV38.1, DV51.6, DV52.1, DV53.4,DV54.7, DV55.1, DV56.12, DV54.7, DV57.4, DV59.3, DV60.3, DV61.2, DV62.5,DV63.1, DV64.3, DV65.5, DV66.1, DV67.9, DV68.2, DV69.6, DV70.1, DV71.1,DV74.4, DV75.9, DV76.5, DV77.5, DV78.6, DV79.3, DV82.11, DV82.11 LALA,DV86.2, DV87.1, DV87.1 LALA, DV90.3, DV257.13, DV291.7, DV415.8, andDV470.12 (Beltramello et al., Cell Host Microbe. 2010; 8(3):271-83);human monoclonal antibodies 3-147, 58/5, 2F5, 2G4, 5F9, and 135.3(Dejnirattisai et al., Science. 2010; 328(5979):745-8); mAb 2H12(Midgley et al. J Immunol. 2012; 188(10):4971-9); humanized monoclonalantibody 1A5 (Goncalvez et al., Proc Natl Acad Sci USA. 2007;104(22):9422-7); and human monoclonal antibody 1C19 (Smith et al., MBio.2013; 4(6):e00873-13); or any of the antibodies disclosed in: WO05/056600 by Lai, C. and Purcell, R. (e.g., antibodies 1A5 and 5H2;WO2010/043977 by Lanzavecchia, A. et al.; WO2013/173348 by Dimitrov etal.; US2013/0259871 by Macary et al.; WO 2013/089647 by Fink et al.; WO2013/035345 by Setthapramote et al.; U.S. Pat. No. 8,637,035 byHan-Chung Wu et al.; or WO 2014/025546 by Sasisekharan, R. et al.; or aderivative of any of the aforesaid antibodies (e.g., a human orhumanized form thereof).

Other therapeutic agents that can be used in combination with ananti-dengue antibody described herein also include, but are not limitedto, for example, alpha-glucosidase I inhibitors (e.g., celgosivir asdescribed in Rathore et al., Antiviral Res. 2011; 92(3):453-60);adenosine nucleoside inhibitors (e.g., NITD008 as described in Yin etal., Proc Natl Acad Sci USA. 2009; 106(48):20435-9); inhibitors of NS3and/or its cofactor NS2B (e.g., compounds that block the NS2B bindingpocket within NS3, e.g., [5-amino-1-(phenyl)sulfonyl-pyrazol-3-yl]compounds, as described in Lescar et al., Antiviral Res. 2008;80(2):94-101); RNA-dependent RNA polymerase (RdRp) inhibitors (e.g.,NITD107 as described in Noble et al., J Virol. 2013; 87(9):5291-5);inhibitors of host pyrimidine biosynthesis, e.g., host dihydroorotatedehydrogenase (DHODH) (e.g., NITD-982 and brequinar as described in Wanget al., J Virol. 2011; 85(13):6548-56); inhibitors of viral NS4B protein(e.g., NITD-618 as described in Xie et al., J Virol. 2011;85(21):11183-95); and iminosugars (e.g., UV-4 as described in Perry etal., Antiviral Res. 2013; 98(1):35-43).

Methods of Diagnosis

In some aspects, the present disclosure provides a diagnostic method fordetecting the presence of a dengue virus E protein in vitro (e.g., in abiological sample, such as a blood sample) or in vivo (e.g., in vivoimaging in a subject). The method includes: (i) contacting the samplewith an antibody molecule described herein, or administering to thesubject, the antibody molecule; (optionally) (ii) contacting a referencesample, e.g., a control sample (e.g., a control biological sample, suchas plasma or blood) or a control subject with an antibody moleculedescribed herein; and (iii) detecting formation of a complex between theantibody molecule, and the sample or subject, or the control sample orsubject, wherein a change, e.g., a statistically significant change, inthe formation of the complex in the sample or subject relative to thecontrol sample or subject is indicative of the presence of dengue virusin the sample. The antibody molecule can be directly or indirectlylabeled with a detectable substance to facilitate detection of the boundor unbound antibody. Suitable detectable substances include variousenzymes, prosthetic groups, fluorescent materials, luminescent materialsand radioactive materials, as described above and described in moredetail below.

The term “sample,” as it refers to samples used for detectingpolypeptides includes, but is not limited to, cells, cell lysates,proteins or membrane extracts of cells, body fluids such as blood, ortissue samples.

Complex formation between the antibody molecule and a dengue virusprotein can be detected by measuring or visualizing either the antibodymolecule bound to the dengue virus protein or unbound antibody molecule.Any suitable detection assays can be used, and conventional detectionassays include an enzyme-linked immunosorbent assays (ELISA), aradioimmunoassay (RIA) or tissue immunohistochemistry. Alternative tolabeling the antibody molecule, the presence of a dengue virus proteincan be assayed in a sample by a competition immunoassay utilizingstandards labeled with a detectable substance and an unlabeled antibodymolecule. In this assay, the biological sample, the labeled standardsand the antibody molecule are combined and the amount of labeledstandard bound to the unlabeled binding molecule is determined. Theamount of a dengue virus protein in the sample is inversely proportionalto the amount of labeled standard bound to the antibody molecule.

EXAMPLES Example 1 Structure-Guided Design of Anti-Dengue AntibodiesEpitope and Template Identification

Various neutralizing epitopes exist in dengue virus E protein dimer.These epitopes include regions, e.g., in EDI, EDII, EDIII, fusion loop,EDI/II hinge, and the EDIII “A” β-strand. EDI and EDII areimmunodominant in humans and can induce weakly neutralizing but highlycross-reactive antibodies. EMI can induce potent neutralizingantibodies. Antibodies directed against the fusion loop, located inEDII; often exhibit cross-serotype reactivity but weak neutralizingactivity. Antibodies directed against the EDIII hinge region can exhibitpotent neutralization but are typically serotype-specific due to lowconservation of the epitope region. Antibodies directed against theEDIII A-strand often exhibit high potency due in part to the greateraccessibility of this region to antibodies, but often have limitedcross-serotype reactivity.

To engineer a broadly reactive and highly potent neutralizing antibodyto dengue virus, mouse mAb 4E11 was identified as a template antibody,as it binds to the EDIII A-strand epitope region and exhibits strongneutralization to DENV-1, DENV-2, and DENV-3 but not DENV-4. 4E11 hasvery low (μM) binding to DENV-4. An approach was utilized to increasemolecular contacts of 4E11 to DENV-4 in order to increase affinity andthereby neutralization potency to DENV-4. To do this, a structural modelof 4E11 with EDIII was generated and then analyzed to identifyserotype-specific binding determinants of 4E11.

Technology and Tool Development

Conventional computational approaches for protein engineering typicallyrely on energetic-based methods. Results from these methods aregenerally highly sensitive to the precise atom locations in a structureor model, and therefore these methods typically require a crystalstructure or similar data of high-resolution and quality of theprotein-protein complex for accurate modeling and beneficial-mutationpredictions. Additionally, conventional energetics-based approaches toprotein engineering typically do not incorporate antibody-specificproperties and knowledge.

A different approach is to use empirical informatics, and specificallyresidue pairwise propensity methods (Tharakaraman K. et al. (2013) ProcNatl Acad Sci USA. 23; 110(17):E1555-64). Engineering of a broadlycross-reactive antibody to dengue virus with broad-spectrum activity andincreased in vivo potency was performed by evaluating the pairwisepropensity (“fitness”) for paratope residues given the epitopeenvironment. This fitness metric is based on empirical data ofantibody-antigen structures and is less sensitive to precise atomlocations as compared to other approaches. Thus, this approach can beeffectively used to enhance accuracy of antibody-antigen computationalmolecular docking and to augment prediction of affinity-enhancingmutations and identification of positions for affinity maturation.

Application: Engineer Template mAb for pM-nM Binding to all Four DENVSerotypes

A 4E11-EDIII structural model was generated and affinity-enhancingmutations/positions were predicted. For example, individual mutationswere predicted at specific sites and positions were selected for thecreation of rational, focused combinatorial libraries. As shown in FIG.23, the resulting humanized antibody-D88 demonstrates, relative toantibody 4E11, a 10,000-fold affinity gain to DENV-4 with concurrentimproved affinity to DENV1-3.

Comparison of mAb A11 with mAb 4E11

A comparison between anti-dengue antibodies 4E11 (which antibody isdescribed in Thullier et al., J Biotechnol. 1999 Apr. 15;69(2-3):183-90) and A11 (4E5A) is provided in Tharakaraman et al., ProcNatl Acad Sci USA. 2013; 110(17):E1555-64. 4E5A has five point mutationsrelative to 4E11, at A55E (VH), R31K (VL), N57E (VL), E59Q (VL), andS60W (VL) (Tharakaraman et al., 2013, Proc Natl Acad Sci USA. 2013;110(17):E1555-64).

Example 2 Del26 Improves DV-4 Neutralization Activity

B11 is an anti-dengue antibody with a heavy chain deletion of S26 inframework 1 (FR1) relative to A11 (4E5A). 4G2 is a control anti-dengueantibody. The neutralization activity of each antibody for EDIII fromfour dengue virus serotypes was tested by a Focus ReductionNeutralization test (FRNT).

The focus reduction neutralization test detects foci formed when denguevirus infects host Vero cells. Briefly, dilutions of antibody are mixedwith an equal volume of diluted virus, and the mixture is transferred toVero cell monolayers, and foci are detected. Data are expressed as therelative infectivity. The FRNT₅₀ represents the concentration ofantibody required to achieve 50% virus neutralization. A more detailedprotocol for the focus neutralization reduction test can be found inTharakaraman et al., 2013, Proc Natl Acad Sci USA. 2013;110(17):E1555-64.

The four graph panels of FIG. 2 show the neutralization activities ofeach antibody against representative strains from dengue virusserotypes. FIG. 3 is a repeated assay of each antibody against denguevirus serotype DV-4. The results are summarized in the table at thebottom of FIG. 2, which shows the IC50 of each antibody against thevirus (in μ/ml).

Unexpectedly, the deletion of one amino acid in the framework 1 regionconfers improved DV-4 neutralization activity on B11. More specifically,compared to A11 which has an IC50 of 4.0-17.6 μg/mL, the antibody B11improves neutralization of DV-4, achieving an IC50 of 0.50-1.4 μg/mL.These results indicate that B11 achieves an about 8 to about 12-foldlower IC50 than A11. Based on B11's superior neutralization activity,humanized variants of B11 were created.

Example 3 Humanized Anti-Dengue Antibodies

FIG. 4 describes some humanized antibodies related to A11 to B11.Various humanization frameworks were tested. The rightmost four columnsshow the affinity of each antibody for EDIII of dengue virus serotypesDV-1, DV-2, DV-4, and DV-4. An A98V mutation in the heavy chain FW3(compare antibody B48+A98V to antibody B48) improved binding to DV-4 byabout 5-fold, while retaining or improving binding to the otherserotypes.

Example 4 Back-Mutations of Humanized Anti-Dengue Antibodies

To improve antibody affinity for EDIII, especially in DV-4, variousback-mutations were made to the heavy chain N-terminus of selectedhumanized antibodies. FIG. 5 shows that D48, which is a full mousereversion of the N-terminus, has an about two-fold improvement inaffinity relative to an antibody with a fully humanized N-terminus. Inother cases, back-mutation resulted in a similar, slightly lower, orslightly higher affinity for EDIII-DV-4.

The rightmost column in the upper and lower table of FIG. 5 shows thathumanized antibodies' affinity for DV-4 are between 7.494 and 26.89 nM.This retention of binding activity indicates that there is a fair amountof tolerance for mutation in the N-terminal region, e.g., in positions1-6 of the heavy chain.

Example 5 Improvement in Antibody Affinity Through a combination ofAffinity-Enhancing Mutations

To further improve the affinity of the humanized antibodies for DV-4,various affinity-enhancing mutations were tested alone or incombination. In FIG. 6, the following mutations were tested: T33V,del26, G27A, G27Y, F28W, F28G, F28A, and F28Y, all in the VH. Del26 andT33V together (in antibody D88) was found to improve affinity forEDIII-DV-4 by about 4-fold compared to the T33V mutation alone (inantibody C88). The double del26 and T33V mutation found in antibody D88also improves affinity over an antibody having the del26 mutation alone.This additive or synergistic improvement in binding was unexpected.

From FIG. 6, it is also apparent that several mutations can be combinedwithout reducing affinity or with only a modest reduction in affinity(e.g., F28W and T33V in antibody D118, G27A and T33V in antibody D98,G27Y and T33V in antibody D148, G27Y and F28W and T33V in antibody D108,and G27A and F28W and T33V in antibody D128). This experiment indicatesthat the antibody has some tolerance for substitutions at positions 27and 28.

Next, mutations at position 98 in the VH were tested in combination withother mutations. The original humanized sequence has A at position 98.FIG. 7 shows that 98V improves binding to DV-4 about two-fold relativeto 98S or 98A, in the context of a T33V mutation (see top three rows ofthe table). The bottom three rows of the table show that mutations toposition 98 do not have a strong effect in the context of the doublemutation del26+T33V. Accordingly, the antibody molecule has sometolerance to mutations to residue 98.

Example 6 Additional Binding Studies on Humanized Anti-Dengue Antibodies

Several humanized anti-dengue antibodies were tested for their abilityto bind EDIII from dengue virus serotypes DV-1, DV-2, DV-3, and DV-4.FIG. 8 shows the antibodies' affinity as determined by a competitionELISA assay, and FIG. 9 shows antibodies' affinity as. determined bySPR. While all the humanized antibodies have strong affinity (e.g., allare less than or equal to 24.35 nM and many are in the sub-nanomolarrange), some antibodies have particularly strong binding. For example,C88 and D88 bind to DV-1 with about 20-fold greater affinity than C98does. All three antibodies of FIG. 9 have about equal affinity for DV-2and for DV-3. Of the antibodies in FIG. 9, D88 has the strongestaffinity for DV-4.

Genetic and antigen diversity of dengue virus exists not only betweenserotypes but also within serotypes. To investigate more completely thebreadth of binding by anti-dengue virus antibodies, a set of analyseswere performed to identify a panel of strain sequences that furthercaptures dengue virus sequence diversity, specifically in EDIII.First, >3,500 E protein sequences of dengue virus isolates from NCBIGenBank were analyzed for amino acid diversity in EDIII. From thisanalysis, a set of 21 strain EDIII sequences were identified andselected to more completely represent dengue virus diversity. Morespecifically, those sequences represent all genotypes having evidence ofrecent circulation and recent isolates from major endemic regions(clinically relevant strains). Challenging isolates, e.g., those havingpositions of diversity near or in the epitope region, were selectedwhenever possible. Additionally, prototypic strains were included. Thephylogenetic relationship of the EDIII amino acid sequences of theselected panel of 21 dengue virus isolates is summarized in FIG. 10A.The results of antibody D88 binding to a panel of EDIII proteins oftwenty-one diverse dengue strains are shown in FIG. 10B. As shown inFIG. 10B, antibody D88 exhibits full spectrum binding to dengue viruses.

Example 7 Humanized Anti-Dengue Antibodies Bind to EDIII From a Numberof Strains of Serotypes DV-2, DV-3, and DV-4

D88 (FIG. 11, rightmost column) and C88 (second column from the right)were tested for the ability to bind EDIII isolated from various strainsof dengue virus. The serotype and geographical location of the strainare indicated in the leftmost two columns. In general, the antibodiesdisplayed a good breadth of binding, indicating that they are likelyeffective against a wide range of dengue virus strains.

Example 8 Humanized Anti-Dengue Antibodies Neutralize Dengue Virus in aFocus Reduction Neutralization Test

Humanized antibodies have the capacity to neutralize DV-1, DV-2, DV-3and DV-4 in a focus reduction neutralization test. The assay isdescribed above in Example 2. FIGS. 12, 13, and 14 show the results ofthese experiments with representative DV-3 and DV-4 strains. Humanizedantibodies are effective in neutralizing dengue virus in this assay. Forinstance, A48, C98, and D88 achieve a lower EC50 for DV-4 than the mouseanti-dengue antibody A11 (FIG. 12). FIG. 13 shows that antibodies D88,D188, and D128 have an EC50 in the range of 426-506 ng/ml for thisstrain of DV-3. In addition, D88, D188, and D128 had a lower EC50 forDV-4 than the mouse anti-dengue antibody A11 (FIG. 14). Humanizedantibodies also have the capacity to neutralize DV-1 and DV-2 (data notshown). It was unexpected that humanized antibodies would achieve ahigher activity than a mouse anti-dengue antibody. Based at least inpart, on binding data shown herein and sequence analysis of known dengueisolates, humanized anti-dengue antibody molecules as described hereinare likely to have broad neutralization capacity across all serotypesand genotypes with those strains listed herein representatives of thediversity of Dengue virus.

Example 9 Thermal Stability of Humanized Antibodies was Tested byThermal Shift Analysis

Selected humanized antibodies were assayed for stability by thermalshift analysis using the reagent Sypro Orange. Results of theexperiments are shown in FIGS. 15A, 15B, and 15C. In general, thehumanized antibodies showed good stability, often having a Tm betweenabout 64 and 68.

Example 10 Mouse Models for Dengue Virus

The antibody molecules described herein can be tested for efficacy in ananimal model. One such model, the AG129 mouse model, is described inTharakaraman et al., 2013, Proc Natl Acad Sci USA. 2013;110(17):E1555-64. The AG129 mouse strain, which lacks both type-I andtype-II interferon receptors, is a commonly used animal model thatreplicates some of the disease manifestations observed in clinical casesof dengue, including viremia and other signs of disease. Infected AG129mice may experience neurological impairment associated with DENVreplication in the brain, which is only very rarely observed in humanpatients infected with the virus. This often leads to death of infectedAG129 mice around 18 days after virus challenge (Stein, D. A., et al., JAntimicrob Chemother, 2008. 62(3): p. 555-65.; Johnson A J, Roehrig J T.J Virol. 1999 January; 73(1):783-6. Despite the lack of similarities tohuman infection, this model is useful in evaluation of antiviraltreatments and can be used in proof of principle studies. Briefly, theAG129 (which is deficient in IFN-α/β and IFN-γ receptors) mouse ischallenged with dengue virus, and a candidate therapeutic antibodymolecule is administered. Typically, viremia (virus titer in a bloodsample) is the endpoint of the experiment. Viremia can be measured,e.g., with quantitative RT-PCR. In this study, viremia, weight change,and survival were used as disease signs after infection with the NewGuinea C (NGC) strain of DENV-2. The results shown in this Exampledemonstrate a strong in vivo inhibitory activity of antibody D88 in aDENV-2 mouse model.

Animals were block-randomized according by cage to groups, with 10included in each. Animals were treated with antibody D88, an irrelevantisotype-matched control mAb (CmAb), or placebo one day prior to viruschallenge. A group of mice, which were not challenged with virus, weretreated with D88 and served as toxicity controls. A group of mice wasalso included as normal controls, and were not treated or challengedwith virus to monitor handling and caging techniques for effects on theimmunocompromised AG129 mice. A 10⁻¹ dilution (10^(6.8) CCID₅₀ (cellculture infectious dose 50%)/ml) of the virus was prepared in minimalessential media. Mice were injected intraperitoneally (i.p.) with 0.4 mlof the diluted virus (10^(6.4) CCID₅₀/animal). Mortality was observeddaily for 31 days. Mice were weighed on day 0 and every other daybeginning at 1 dpi (day post infection). Serum was collected from allanimals on 3 dpi for quantification of viremia by qRT-PCR.

FIG. 16 shows the survival percentage of mice administered D88 (25mg/kg), D88 (5 mg/kg), CmAb, or PBS, after infection with dengue virus.As shown in FIG. 16, about 90% of the mice treated with 25 mg/kg of D88and about 60% of the mice treated with 5 mg/kg of D88 survived until Day31, whereas control mice treated with CmAb or PBS all died on or beforeDay 17.

FIG. 17 shows the mean weight change of mice administered D88 (25mg/kg), D88 (5 mg/kg), CmAb, or PBS, after infection with dengue virus.Mice treated with D88 (25 mg/kg) or PBS but not infected with denguevirus (sham) were also tested. As shown in FIG. 17, mice administeredantibody D88 had no significant weigh change even 31 days postinfection, whereas mice administered CmAb or PBS had significant weightloss at Day 15. As a control, mice treated with D88 or PBS but notinfected with dengue virus did not exhibit apparent weight change.

FIG. 18 shows the viremia titer in mice administered D88 (25 mg/kg), D88(5 mg/kg), CmAb, or PBS, after infection with dengue virus. The resultsare expressed as extrapolated PFU per ml and genome copy equivalents(GCE)/mL. Significance with a p value <0.001 is shown by the symbols***. As shown in FIG. 18, mice administered CmAb (isotype control) orPBS had higher viremia titer compared to mice treated with 25 mg/kg ofD88 or 5 mg/kg of D88.

These results demonstrate that a single systemic administration ofantibody D88 resulted in rapid reduction of circulating viral titers.Antibody D88 provided strong protection, with 9/10 and 6/10 animals at25 and 5 mg/kg, respectively, surviving DENV-2 lethal challenge.Antibody D88 demonstrated a significant and dose-dependent reduction inviral titer on day 3 post-infection, the day of peak viremia.

Example 11 Protection Against ADE in vivo

The antibody molecules described herein can be tested for efficacy inanimal models. One such model, antibody-enhanced severe dengue virusinfection in AG129 mice, is described in Balsitis et al., 2010 (Lethalantibody enhancement of dengue disease in mice is prevented by Fcmodification, PLoS Pathogens, 2010 Feb. 12; 6(2):e1000790). Briefly,AG129 mice are administered dengue virus enhancing antibody (e.g., DV1antiserum or 4G2 monoclonal antibody) 1 da_(y) prior to challenge withdengue virus (e.g., D2S10). Candidate antibody molecule is administered1 day prior to challenge (prophylaxis) or 1 or 2 days after challenge(therapeutic). Typically, mortality is the endpoint of the experiment;viremia and inflammatory cytokine (e.g., TNF-α) levels in the serum mayalso serve as endpoints.

Example 12 Neutralization of Dengue Virus Serotypes Propagated in Insectand Mammalian Cells

The neutralization activity of humanized antibodies against four denguevirus serotypes was examined by a Focus Reduction Neutralization test(FRNT) as described above in Example 2. Four dengue virus serotypes(DENV-1, DENV-2, DENV-3 and DENV-4) were propagated in either insectcells or mammalian cells and were used to infect host Vero (monkey)cells. FIGS. 16-18 show the results of these experiments withrepresentative DENV-1-4 strains. Data are expressed as the relativeinfectivity. For example, the EC₅₀ or FRNT₅₀ values represent theconcentrations of antibody required to achieve 50% virus neutralization.As shown in FIGS. 16-18, the tested antibodies are effective inneutralizing DENV-1, DENV-2, DENV-3 and DENV-4 strains propagated ininsect and mammalian cells.

FIG. 19 shows that antibody D88 neutralized DENV-1, DENV-2, DENV-3 andDENV-4 strains propagated in C6/36 insect cells. The results aresummarized in the table at the bottom of FIG. 19, which shows the EC₅₀values against representative DENV-1, DENV-2, DENV-3 and DENV-4 strains(in ng/ml). DENV-1 strain Hawaii/1944, DENV-2 strain New Guinea/1944(NGC), DENV-3 strain Philippines/1956 (H87), and DENV-4 strainMexico/1997 (BC287/97) were tested.

FIG. 20 shows that antibody D88 neutralized DENV-1, DENV-2, DENV-3 andDENV-4 strains propagated in Vero cells. The results are shown as theFRNT₅₀ values against representative DENV-1, DENV-2, DENV-3 and DENV-4strains (in ng/ml). DENV-1 strain Hawaii/1944, DENV-2 strain NewGuinea/1944 (NGC), DENV-3 strain Philippines/1956 (H87), and DENV-4strain Mexico/1997 (BC287/97) were tested.

FIG. 21 shows that antibodies D88 and A11 neutralized DENV-4 strain H241propagated in Vero cells. The results are summarized in the table at thebottom of FIG. 21, which shows the EC₅₀ values of the antibodies againstDENV-4 strain H241 (in ng/ml).

These results demonstrate that antibody D88 potently neutralized allfour DENV serotypes with EC50 values of <1 μg/ml. Antibody D88efficiently neutralized the challenging DENV-4 strain H241, to which itbound with 100 nM affinity.

Example 13 Mosquito Models for Dengue Virus to Evaluate InhibitoryActivity of Antibodies

The antibody molecules described herein can be tested for efficacy in amosquito model. Dengue virus is a mosquito transmitted RNA virus.Certain dengue virus can develop in vivo fitness advantage, which mayresult in higher probability of human-to-mosquito transmission (Vu etal., PLoS Negl Trop Dis. 2010; 4(7):e757). To establish a mosquito modelto evaluate inhibitory activity of antibodies against dengue virus,blood containing virus is mixed with antibody at various dilutions andincubate at 37° C. for 30 minutes. Antibody-spiked blood is added to amosquito feeder and mosquitoes are fed for about 1 hour. Mosquitoes arecold-anaesthetized and engorged ones are selected. Mosquitoes' abdomensare collected at day 7 after blood feeding. Viral load can be measuredby qRT-PCR. The proportion of mosquitoes with abdomen infection can becalculated as the number of infected abdomens divided by the totalnumber of abdomens tested by PCR.

Example 14 HP-SEC Evaluation of Anti-Dengue Antibodies

High performance size exclusion chromatography (HP-SEC) was performed toevaluate aggregation propensity of anti-dengue antibodies under native,non-stressed conditions. This method allows for discrimination ofantibody dimers and aggregates from monomers. Dimers and aggregates maylead to increased risk of immunogenicity.

In this study, antibodies were purified to 1 mg/ml and evaluated by asize exclusion column, e.g., Phenomenex BioSep s3000, using PBS as amobile phase with a flow rate of 1 ml/min.

FIG. 22 shows a representative chromatogram of antibody D88, whichdisplays greater than 98% of antibody (purified only by Protein Achromatography) present as monomer. As summarized in the table at thebottom of FIG. 22, 99.15% of antibody A48, 98.37% of antibody C88, and99.59% of antibody D88, were present as monomers in the samples.

INCORPORATION BY REFERENCE

All publications, patents, and accession numbers mentioned herein arehereby incorporated by reference in their entirety as if each individualpublication or patent was specifically and individually indicated to beincorporated by reference.

EQUIVALENTS

While specific embodiments of the compositions and methods herein havebeen discussed, the above specification is illustrative and notrestrictive. Many variations of the invention will become apparent tothose skilled in the art upon review of this specification and theclaims below. The full scope of the invention should be determined byreference to the claims, along with their full scope of equivalents, andthe specification, along with such variations.

1.-24. (canceled)
 25. A method of neutralizing dengue virus, the methodcomprising contacting the dengue virus with an antibody molecule capableof binding to the dengue virus, wherein the antibody molecule comprises:(a) a heavy chain immunoglobulin variable region segment comprising: aCDR1 comprising the sequence DVYMS (SEQ ID NO: 3), a CDR2 comprising thesequence RIDPENGDTKYDPKLQG (SEQ ID NO: 4), and a CDR3 comprising thesequence GWEGFAY (SEQ ID NO: 5); and (b) a light chain immunoglobulinvariable region segment comprising: a CDR1 comprising the sequenceRASENVDKYGNSFMH (SEQ ID NO: 6), a CDR2 comprising the sequence RASELQW(SEQ ID NO: 7), and a CDR3 comprising the sequence QRSNEVPWT (SEQ ID NO:8), thereby neutralizing dengue virus. 26.-30. (canceled)
 31. The methodof claim 25, wherein the dengue virus is of serotype DV-1, DV-2, DV-3,and DV-4.
 32. A method of inhibiting dengue virus, the method comprisingadministering to a subject in need thereof an antibody molecule capableof binding to the dengue virus, wherein the antibody molecule comprises:(a) a heavy chain immunoglobulin variable region segment comprising: aCDR1 comprising the sequence DVYMS (SEQ ID NO: 3), a CDR2 comprising thesequence RIDPENGDTKYDPKLQG (SEQ ID NO: 4), and a CDR3 comprising thesequence GWEGFAY (SEQ ID NO: 5); and (b) a light chain immunoglobulinvariable region segment comprising: a CDR1 comprising the sequenceRASENVDKYGNSFMH (SEQ ID NO: 6), a CDR2 comprising the sequence RASELQW(SEQ ID NO: 7), and a CDR3 comprising the sequence QRSNEVPWT (SEQ ID NO:8), thereby inhibiting dengue virus.
 33. The method of claim 32, whereinthe antibody molecule neutralizes dengue virus serotypes DV-1, DV-2,DV-3, and DV-4.
 34. The method of claim 32, wherein the subject isinfected with dengue virus.
 35. The method of claim 32, wherein theantibody molecule is administered to the subject prior to the subjectbeing exposed to dengue virus.
 36. The method of claim 32, wherein theantibody molecule is administered to the subject prior to onset of oneor more manifestations of dengue virus.
 37. The method of claim 32,wherein the antibody molecule comprises a VH FW1 having the sequenceQVQLVQSGAEVKKPGASVKVSCKAGFNIK (SEQ ID NO: 11) or a VH FW1 comprising adeletion of position 26 relative to SEQ ID NO:
 33. 38. The method ofclaim 32, wherein the antibody molecule comprises a VH FW2 having thesequence WVRQAPGQGLEWMG (SEQ ID NO: 84) or WVRQAPEQGLEWMG (SEQ ID NO:85).
 39. The method of claim 32, wherein the antibody molecule comprisesthe VH amino acid sequence of SEQ ID NO:
 1. 40. The method of claim 32,wherein the antibody molecule comprises the VL amino acid sequence ofSEQ ID NO:
 2. 41. The method of claim 32, wherein the antibody moleculecomprises the VH amino acid sequence of SEQ ID NO: 1 and the VL aminoacid sequence of SEQ ID NO:
 2. 42. The method of claim 32, wherein theantibody molecule is a Fab, F(ab′)2, Fv, or a single chain Fv fragment(scFv).
 43. The method of claim 32, wherein the antibody moleculecomprises a heavy chain constant region chosen from IgG1, IgG2, IgG3, orIgG4.
 44. The method of claim 32, wherein the antibody moleculecomprises a light chain constant region chosen from the light chainconstant regions of kappa or lambda.
 45. The method of claim 32, whereinthe antibody molecule contains one or more framework regions derivedfrom a human framework germline sequence.
 46. The method of claim 32,wherein the antibody molecule is capable of binding to dengue virusEDIII (E protein domain III).
 47. The method of claim 46, wherein theantibody molecule is capable of binding to dengue virus EDIII with adissociation constant (K_(D)) of less than about 30 nM.
 48. The methodof claim 46, wherein the antibody molecule is capable of binding todengue virus serotype DV-4 EDIII with a dissociation constant (K_(D)) ofless than about 10 nM.
 49. The method of claim 46, wherein the antibodymolecule is capable of binding to DV-3 or DV-4 EDIII domain with atleast a 6-fold greater affinity than antibody A11 or antibody 4E11. 50.The method of claim 32, wherein the antibody molecule is capable ofbinding to a dengue virus strain selected from DENV-4 BC2, DENV-4-Sing,DENV-4 NC, DENV-4 Phil, DENV-3 Sing, DENV-3 Nic, DENV-3 H87, DENV-2Peru, DENV-2 Sing, DENV-2 NGC, DENV-1 Hawaii/1944, DENV-2 NewGuinea/1944 (NGC), DENV-3 Philippines/1956 (H87), DENV-4 Mexico/1997(BC287/97), and DENV-4 H241, with at least 2-fold greater affinity thanantibody A11 or antibody 4E11.
 51. The method of claim 32, wherein theantibody molecule is capable of neutralizing dengue virus in a focusreduction neutralization test.
 52. The method of claim 32, wherein theantibody molecule is capable of neutralizing dengue virus with an IC50that is at least 2-fold lower than antibody A11 or antibody 4E11 in afocus reduction neutralization test.
 53. The method of claim 32, furthercomprising administering an anti-viral agent to the subject.
 54. Themethod of claim 53, wherein the anti-viral agent is chosen from one ormore of balapiravir, chloroquine, celgosivir, ivermectin, or Caricafolia.
 55. The method of claim 53, wherein the anti-viral agent is ananti-dengue antibody chosen from one or more of any of the antibodieslisted in Table 1, 4E11, 14c10 (HM14c10), 1F4, 2D22, 5J7, DV1.1, DV1.6,DV3.7, DV4.4, DV5.1, DV6.1, DV7.5, DV8.1, DV10.16, DV13.4, DV13.8,DV14.5, DV14.5, DV15.7, DV16.5, DV16.8, DV17.6, DV18.21, DV18.4, DV19.3,DV20.1, DV21.1, DV21.5, DV22.3, DV22.3 LALA, DV23.13, DV25.5, DV27.2,DV28.1, DV28.8, DV34.4, DV35.3, DV38.1, DV51.6, DV52.1, DV53.4, DV54.7,DV55.1, DV56.12, DV54.7, DV57.4, DV59.3, DV60.3, DV61.2, DV62.5, DV63.1,DV64.3, DV65.5, DV66.1, DV67.9, DV68.2, DV69.6, DV70.1, DV71.1, DV74.4,DV75.9, DV76.5, DV77.5, DV78.6, DV79.3, DV82.11, DV82.11 LALA, DV86.2,DV87.1, DV87.1 LALA, DV90.3, DV257.13, DV291.7, DV415.8, DV470.12,3-147, 58/5, 2F5, 2G4, 5F9, 135.3, 2H12, 1A5, or 1C19.
 56. The method ofclaim 53, wherein the anti-viral agent is chosen from one or more of analpha-glucosidase I inhibitor, an adenosine nucleoside inhibitor, aninhibitor of NS3 and/or its cofactor NS2B, an RNA-dependent RNApolymerase (RdRp) inhibitor, an inhibitor of host pyrimidinebiosynthesis, an inhibitor of viral NS4B protein, or an iminosugar. 57.The method of claim 56, wherein the alpha-glucosidase I inhibitor iscelgosivir, the adenosine nucleoside inhibitor is NITD008, the inhibitorof NS3 and/or its cofactor NS2B is a[5-amino-1-(phenyl)sulfonyl-pyrazol-3-yl] compound, the RNA-dependentRNA polymerase (RdRp) inhibitor is NITD107, the inhibitor of hostpyrimidine biosynthesis is an inhibitor of host dihydroorotatedehydrogenase (DHODH) selected from NITD-982 or brequinar, the inhibitorof viral NS4B protein is NITD-618, or the iminosugar is UV-4.
 58. Themethod of claim 32, further comprising administering a vaccine to thesubject.
 59. The method of claim 32, wherein administration isparenteral or intravenous.